Alpha2 subunit of prolyl 4-hydroxylase

ABSTRACT

The present invention relates to novel isoforms of the α subunit of prolyl 4-hydroxylase, polynucleotide sequences encoding these novel proteins, and methods for making such proteins.

[0001] This application is a continuation of U.S. application Ser. No. 09/686,322, filed Oct. 10, 2000, which is a continuation of U.S. application Ser. No. 09/196,581, filed Nov. 20, 1998, which is a divisional of U.S. application Ser. No. 08/633,879, filed Apr. 10, 1996, now U.S. Pat. No. 5,928,922, issued Jul. 27, 1999.

1. INTRODUCTION

[0002] The present invention relates to the identity and characterization of novel α subunits of prolyl 4-hydroxylase, variants thereof; polynucleotide sequences which encode the novel α2 subunits of prolyl 4-hydroxylase, and methods for using and making such novel polynucleotides and polypeptides. The present invention also relates to the recombinant production of active: (1) prolyl 4-hydroxylase, or variants thereof, and (2) collagen, comprising the use of the novel human a subunit of prolyl 4-hydroxylase of the present invention.

[0003] The present invention more specifically relates to polynucleotides encoding a novel isoform of the a subunit of prolyl 4-hydroxylase, designated the “α2 subunit,” and derivatives thereof, methods for producing such isoforms or related derivatives and the use of these proteins and polynucleotides in the production of recombinant collagen.

2. BACKGROUND

[0004] General Information Regarding Collagen.

[0005] Collagen fibrils, proteoglycan aggregates and glycoproteins are critical components of the cartilage extracellular matrix that, collectively, resist compression and the tensile and shear forces that are generated during articulation. Heineg.ang.rd and Oldberg (1989) FASEB J. 3:2042-2051; Mayne and Brewton (1993) Cartilage Degradation: Basic and Clinical Aspects (Woessner, J. F. and Howell, D. S., eds.) Marcel Dekker, Inc., New York, pp. 81-108. Mutations in cartilage matrix genes or the genes that encode the enzymes that affect the biosynthesis, assembly or interactions between these various matrix components may contribute to degradation of the cartilage matrix and the loss of normal cartilage function.

[0006] The Role of Prolyl 4-Hydroxylase in the Production of Collagen.

[0007] Prolyl 4-hydroxylase plays a crucial role in the synthesis of all collagens. Specifically, the enzyme catalyzes the formation of 4-hydroxyproline in collagens and related proteins by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. These 4-hydroxyproline residues are essential for the folding of newly synthesized collagen polypeptide chains into triple-helical molecules.

[0008] The vertebrate prolyl 4-hydroxylase is an α₂β₂ tetramer in which the a subunits contribute to most parts of the catalytic sites. See, Kivirikko et al. (1989) FASEB J. 3, 1609-1617; Kivirikko et al. (1990) Ann. N.Y. Acad. Sci. 580, 132-142; Kivirikko et al. (1992) Post Translational Modifications of Proteins (Harding, J. J. and Crabbe, M. J. C., eds.) CRC, Boca Raton, Fla., pp. 1-51. The β subunit has been cloned from many sources (id.; see also, Noiva and Lennatz (1992) J. Biol. Chem. 267:6447-49; Freedman et al. (1994) Trends Biochem. Sci. 19:331-336) and has been found to be a highly unusual multifunctional polypeptide that is identical to the enzyme protein disulfide-isomerase (Pihlajaniemi et al. (1987) EMBO J. 6:643-649; Kojvu et al. (1987) J. Biol. Chem. 262:6447-49), a cellular thyroid hormone-binding protein (Cheng et al. (1987) J. Biol. Chem. 262:11221-27), the smaller subunit of the microsomal triacylglycerol transfer protein (Wetterau et al. (1990) J. Biol. Chem. 265:9800-07), and an endoplasmic reticulum luminal polypeptide which uniquely binds to various peptides (Freedman, supra; Noiva et al. (1991) J. Biol. Chem. 266:19645-649; Noiva et al. (1993) J. Biol. Chem. 268:19210-217).

[0009] A catalytically important α subunit, designated the α1 subunit, has been cloned from human (Helaakoski et al. (1989) Proc. Natl. Acad. Sci. USA 86:4392-96), chicken (Bassuk et al. (1989) Proc. Natl. Acad. Sci. USA 86:7382-886) and Caenorhabditis elegans (Veijola et al. (1994) J. Biol. Chem. 269:26746-753), and its RNA transcripts have been shown to undergo alternative splicing involving sequences encoded by two consecutive, homologous 71-bp exons (Helaakoski, supra; Helaakoski et al. (1994) J. Biol. Chem. 269:27847-854). A second a subunit, designated the a² subunit has been previously obtained from mouse. Helaakoski et al. (1995) Proc. Natl. Acad. Sci. USA 92:4427-4431.

3. SUMMARY OF THE INVENTION

[0010] The present invention is directed to the cloning and characterization of human α-subunit isoforms of prolyl 4-hydroxylase. More specifically, the present invention relates to human subunit isoforms of the a subunit of prolyl 4-hydroxylase designated the α2 subunit, and the polynucleotide sequences which encode them. Also described herein are methods for producing the α2 subunit of prolyl 4-hydroxylase, prolyl 4-hydroxylase and collagen, wherein said prolyl 4-hydroxylase is comprised of the α2 subunit of the present invention and said collagen is processed into its proper form by such prolyl 4-hydroxylase. In accordance with the invention, any nucleotide sequence that encodes the amino acid sequence of claimed α2 subunit of prolyl 4-hydroxylase can be used to generate recombinant molecules that direct the expression of human prolyl 4-hydroxylase.

[0011] The present invention is further directed to the use of the coding sequence for the α2 subunit of prolyl 4-hydroxylase to produce an expression vector which may be used to transform appropriate host cells. The host cells of the present invention are then induced to express the coding sequence and thereby produce the α2 subunit of prolyl 4-hydroxylase, or more generally, in combination with the p subunit, prolyl 4-hydroxylase.

4. DETAILED DESCRIPTION

[0012] The present invention relates to human α2 subunits of prolyl 4-hydroxylase and nucleic acid sequences encoding these α2 subunits of the prolyl 4-hydroxylase and derivatives thereof. In accordance with the invention, any nucleotide sequence which encodes the amino acid sequence of claimed human α2 subunit of prolyl 4-hydroxylase can be used to generate recombinant molecules which direct the expression of prolyl 4-hydroxylase. Also within the scope of the invention are methods of using and making these α2 subunits of prolyl-4hydroxylase.

[0013] a. Definitions

[0014] The term “α2 subunit of prolyl-4-hydroxylase” refers to isoforms of the a subunit of prolyl 4-hydroxylase, as encoded by a single gene as set forth at SEQ ID NO: 3, and genes which contain conservative substitutions thereto.

[0015] “Active human prolyl 4-hydroxylase” refers to a protein complex comprising a prolyl 4-hydroxylase α₂β₂ tetramer, and may be recombinantly produced.

[0016] The phrase “stringent conditions” as used herein refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5× SSC (0.75 M NaCl, 0.075 M Sodium citrate), 5× Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS.

[0017] The term “purified” as used in reference to prolyl 4-hydroxylase denotes that the indicated molecules are present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. The term “purified” as used herein preferably means at least 95% by weight, more preferably at least 99.8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons can be present).

[0018] The term “isolated” as used herein refers to a protein molecule separated not only from other proteins that are present in the source of the protein, but also from other proteins, and preferably refers to a protein found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms “isolated” and “purified” do not encompass proteins present in their natural source.

b. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIGS. 1A and 1B (FIG. 1A, FIG. 1B) set forth the nucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) for the α(2) subunit of mouse prolyl 4-hydroxylase.

[0020]FIGS. 2A, 2B, and 2C (FIG. 2A, FIG. 2B, FIG. 2C) set forth the nucleotide (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) for the α(2) subunit of human prolyl 4-hydroxylase, as derived from cDNA clones.

[0021]FIG. 3 (FIG. 3) sets forth the nucleotide (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:6) for EXON 2 (as identified in FIG. 2) and flanking intron sequences.

[0022]FIG. 4 (FIG. 4) sets forth the nucleotide (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO:8) for EXON 3 (as identified in FIG. 2) and flanking intron sequences.

[0023]FIG. 5 (FIG. 5) sets forth the nucleotide (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO:10) for EXON 4 (as identified in FIG. 2) and flanking intron sequences.

[0024]FIG. 6 (FIG. 6) sets forth the nucleotide (SEQ ID NO:11) and deduced amino acid sequence (SEQ ID NO:12) for EXON 5 (as identified in FIG. 2) and flanking intron sequences.

[0025]FIG. 7 (FIG. 7) sets forth the nucleotide (SEQ ID NO:13) and deduced amino acid sequence (SEQ ID NO: 14) for EXON 6 (as identified in FIG. 2) and flanking intron sequences.

[0026]FIG. 8 (FIG. 8) sets forth the nucleotide (SEQ ID NO: 15) and deduced amino acid sequence (SEQ ID NO: 16) for EXON 7 (as identified in FIG. 2) and flanking intron sequences.

[0027]FIGS. 9A, 9B and 9C (FIG. 9A, FIG. 9B, FIG. 9C) set forth the nucleotide (SEQ ID NO:17) and deduced amino acid sequence (SEQ ID NO:18) for EXON 8 (as identified in FIG. 2) and flanking intron sequences.

c. EXPRESSION Of The α2 SUBUNIT OF PROLYL 4-HYDROXYLASE OF THE INVENTION

[0028] (1) Coding Sequences

[0029] In accordance with the invention, polynucleotide sequences which encode a human isoform of the a subunit of prolyl 4-hydroxylase, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of the human α2 subunit of prolyl 4-hydroxylase or its derivatives, and prolyl 4-hydroxylase comprising the α2 subunit of prolyl 4-hydroxylase, or a functional equivalent thereof, in appropriate host cells. Such sequences of an α2 subunit of prolyl 4-hydroxylase, as well as other polynucleotides which selectively hybridize to at least a part of such polynucleotides or their complements, may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc.

[0030] Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of α2 subunit of prolyl 4-hydroxylase proteins. Such nucleic acid sequences include those which are capable of hybridizing to the appropriate α2 subunit of prolyl 4-hydroxylase sequence under stringent conditions.

[0031] Altered nucleic acid sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The nucleic acid product itself may contain deletions, additions or substitutions of amino acid residues within an α2 subunit of the prolyl 4-hydroxylase sequence, which result in a silent change thus producing a functionally equivalent a subunit. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

[0032] The nucleic acid sequences of the invention may be engineered in order to alter the α2 subunit of the prolyl 4-hydroxylase coding sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product. For example, alternative secretory signals may be substituted for the native human secretory signal and/or mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.

[0033] Additionally, when expressing in non-human cells, the polynucleotides encoding the prolyl 4-hydroxylase of the invention may be modified so as to better conform to the codon preference of the particular host organism.

[0034] In an alternate embodiment of the invention, the coding sequence of the α2 subunit of prolyl 4-hydroxylase of the invention could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al. (1980) Nucleic Acids Symp. Ser. 7:215-233; Crea and Horn (1980) Nucleic Acids Res. 9(10):2331; Matteucci and Caruthers (1980) Tetrahedron Letters 21:719; and Chow and Kempe (1981) Nucleic Acids Res. 9(12):2807-2817. Alternatively, the protein itself could be produced using chemical methods to synthesize the desired α2 subunit amino acid sequence at least in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. See, e.g., Creighton (1983) Proteins Structures And Molecular Principles, W.H. Freeman and Co., New York, pp. 50-60. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton (1983) Proteins, Structures and Molecular Principles, W.H. Freeman and Co., New York, pp. 34-49.

[0035] In order to express the α2 subunit of prolyl 4-hydroxylase of the invention, the nucleotide sequence encoding the α2 subunit of prolyl 4-hydroxylase, or a functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.

[0036] (2) Expression Systems

[0037] Methods which are well known to those skilled in the art can be used to construct expression vectors containing an α2 subunit of prolyl 4-hydroxylase coding sequence for prolyl 4-hydroxylase and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination. See, for example, the techniques described in Maniatis et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York and Ausubel et al. (1989) Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York.

[0038] A variety of host-expression vector systems may be utilized to express a coding sequence of an α2 subunit of prolyl 4-hydroxylase. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a coding sequence of an α2 subunit of prolyl 4-hydroxylase; yeast transformed with recombinant yeast expression vectors containing a coding sequence of an α2 subunit of prolyl 4-hydroxylase; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing sequence encoding the α2 subunit of prolyl 4-hydroxylase; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a coding sequence of an α2 subunit of prolyl 4-hydroxylase; or animal cell systems infected with appropriate vectors, preferably semliki forest virus.

[0039] Additionally, the α2 subunit of prolyl 4-hydroxylase of the invention may be expressed in transgenic non-human animals wherein the desired enzyme product may be recovered from the milk of the transgenic animal. The expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines that contain multiple copies of an α2 subunit of prolyl 4-hydroxylase DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.

[0040] In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the α2 subunit of the prolyl 4-hydroxylase expressed. For example, when large quantities of the polypeptides of the invention are to be produced, vectors which direct the expression of high levels of protein products that are readily purified may be desirable. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al. (1983) EMBO J. 2:1791), in which the polypeptide coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid AS-lac Z protein is produced; pIN vectors (Inouye and Inouye (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as proteins with glutathione S-transferase (GST). In general, such proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.

[0041] A preferred expression system is a yeast expression system. In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see Ausubel et al. (1988) Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al. (1987) Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, Acad. Press, New York 153:516-544; Glover (1986) DNA Cloning, Vol. II, IRL Press, Washington, D.C., Ch. 3; Bitter (1987) Heterologous Gene Expression in Yeast, in Methods in Enzymology, (Berger and Kimmel, eds.) Acad. Press, New York 152:673-684; and Strathern et al. (1982) The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Press, Vols. I and II.

[0042] A particularly preferred system useful for cloning and expression of the proteins of the invention uses host cells from the yeast Pichia. Species of non-Saccharomyces yeast such as Pichia pastoris appear to have special advantages in producing high yields of recombinant protein in scaled up procedures. Additionally, a Pichia expression kit is available from Invitrogen Corporation (San Diego, Calif.).

[0043] There are a number of methanol responsive genes in methylotrophic yeasts such as Pichia pastoris, the expression of each being controlled by methanol responsive regulatory regions (also referred to as promoters). Any of such methanol responsive promoters are suitable for use in the practice of the present invention. Examples of specific regulatory regions include the promoter for the primary alcohol oxidase gene from Pichia pastoris AOX1, the promoter for the secondary alcohol oxidase gene from P. pastoris AX02, the promoter for the dihydroxyacetone synthase gene from P. pastoris (DAS), the promoter for the P40 gene from P. pastoris, the promoter for the catalase gene from P. pastoris, and the like.

[0044] Typical expression in Pichia pastoris is obtained by the promoter from the tightly regulated AOX1 gene. See Ellis et al. (1985) Mol. Cell. Biol. 5:1111, and U.S. Pat. No. 4,855,231. This promoter can be induced to produce high levels of recombinant protein after addition of methanol to the culture. By subsequent manipulations of the same cells, expression of genes for the α2 subunit of prolyl 4-hydroxylase of the invention described herein is achieved under conditions where a recombinant collagen protein is adequately hydroxylated by the prolyl 4-hydroxylase of the present invention and, therefore, can fold into a stable helix that is required for the normal biological function of the collagen in forming fibrils.

[0045] Another particularly preferred yeast expression system makes use of the methylotrophic yeast Hansenula polymorpha. Growth on methanol results in the induction of key enzymes of the methanol metabolism, namely MOX (methanol oxidase), DAS (dihydroxyacetone synthase) and FMHD (formate dehydrogenase). These enzymes can constitute up to 30-40% of the total cell protein. The genes encoding MOX, DAS, and FMDH production are controlled by very strong promoters which are induced by growth on methanol and repressed by growth on glucose. Any or all three of these promoters may be used to obtain high level expression of heterologous nucleic acid sequences in H. polymorpha. The nucleic acid sequence encoding a α2 subunit of prolyl 4-hydroxylase of the invention is cloned into an expression vector under the control of an inducible H. polymorpha promoter. If secretion of the product is desired, a polynucleotide encoding a signal sequence for secretion in yeast, such as the S. cerevisiae prepro-mating factor α1, is fused in frame with the coding sequence for the α2 subunit of the prolyl 4-hydroxylase of the invention. The expression vector preferably contains an auxotrophic marker gene, such as URA3 or LEU2, which may be used to complement the deficiency of an auxotrophic host.

[0046] The expression vector is then used to transform H. polymorpha host cells using techniques known to those of skill in the art. An interesting and useful feature of H. polymorpha transformation is the spontaneous integration of up to 100 copies of the expression vector into the genome. In most cases, the integrated DNA forms multimers exhibiting a head-to-tail arrangement. The integrated foreign DNA has been shown to be mitotically stable in several recombinant strains, even under non-selective conditions. This phenomena of high copy integration further adds to the high productivity potential of the system.

[0047] In cases where plant expression vectors are used, the expression of sequences encoding the α2 subunits of the invention may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. (1984) Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al. (1987) EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al. (1984) Science 224:838-843); or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New York, Section VIII, pp. 421-463; and Grierson and Corey (1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

[0048] An alternative expression system which could be used to express the α2 subunit of prolyl 4-hydroxylase of the invention is an insect system. In one such system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Coding sequence for the α2 subunit of prolyl 4-hydroxylase of the invention may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of a α2 subunit of prolyl 4-hydroxylase coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (See, e.g., Smith et al. (1983) J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051.)

[0049] In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, coding sequence for the α2 subunit prolyl 4-hydroxylase of the invention may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the polypeptide in infected hosts. (See, e.g., Logan and Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659.) Alternatively, the vaccinia 7.5 K promoter may be used. (See, e.g., Mackett et al. (1982) Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al. (1984) J. Virol. 49:857-864; and Panicali et al. (1982) Proc. Natl. Acad. Sci. 79:4927-4931.)

[0050] Specific initiation signals may also be required for efficient translation of inserted prolyl 4-hydroxylase coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire polypeptide gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of a coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the α2 subunit of prolyl 4-hydroxylase coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al. (1987) Methods in Enzymol. 153:516-544).

[0051] One preferred expression system for the recombinant production of the α2 subunit of prolyl 4-hydroxylase of the invention is in transgenic non-human animals, wherein the desired polypeptide may be recovered from the milk of the transgenic animal. Such a system is constructed by operably linking the DNA sequence encoding the α2 subunit of the invention to a promoter and other required or optional regulatory sequences capable of effecting expression in mammary glands. Likewise, required or optional post-translational enzymes may be produced simultaneously in the target cells, employing suitable expression systems, as disclosed in, inter alia, U.S. application, Ser. No. 8/037,728, operable in the targeted milk protein producing mammary gland cells.

[0052] For expression in milk, the promoter of choice would preferably be from one of the abundant milk-specific proteins, such as alpha S1-casein, or β-lactoglobulin. For example, 5′ and 3′ regulatory sequences of alpha S1-casein have been successfully used for the expression of the human lactoferrin cDNA, and similarly, the β-lactoglobin promoter has effected the expression of human antitrypsin gene fragments in sheep milk producing cells. Wright et al. (1991) Biotechnology 9:830-833. In transgenic goats, the whey acid promoter has been used for the expression of human tissue plasminogen activator, resulting in the secretion of human tissue plasminogen activator in the milk of the transgenics. Ebert et al. (1991) Biotechnology 9:835-838. Using such expression systems, animals are obtained which secrete the polypeptides of the invention into milk. Using procedures well-known by those of the ordinary skill in the art, the gene encoding the desired prolyl 4-hydroxylase chain can simply be ligated to suitable control sequences which function in the mammary cells of the chosen animal species. Expression systems for the genes encoding the α2 subunit of prolyl 4-hydroxylase are constructed analogously.

[0053] Preferably, the prolyl 4-hydroxylase of the invention is expressed as a secreted protein. When the engineered cells used for expression of the proteins are non-human host cells, it is often advantageous to replace the human secretory signal peptide of the prolyl 4-hydroxylase protein with an alternative secretory signal peptide which is more efficiently recognized by the host cell's secretory targeting machinery. The appropriate secretory signal sequence is particularly important in obtaining optimal fungal expression of mammalian genes. For example, in methylotrophic yeasts, a DNA sequence encoding the in-reading frame S. cerevisiae α-mating factor pre-pro sequence may be inserted at the amino-terminal end of the coding sequence. The αMF pre-pro sequence is a leader sequence contained in the αMF precursor molecule, and includes the lys-arg encoding sequence which is necessary for proteolytic processing and secretion (see, e.g., Brake et al. (1984) Proc. Natl. Acad. Sci. USA, 81:4642).

[0054] Also preferably, the α2 subunits of prolyl 4-hydroxylase of the present invention are co-expressed by the host cell with a β subunit of prolyl 4-hydroxylase and/or collagen, as described generally in PCT Application No. PCT/US92/09061 (WO 93/07889), such that an α₂β₂ prolyl 4-hydroxylase tetramer is formed and this enzyme catalyzes the formation of 4-hydroxyproline in the expressed collagen.

[0055] Alternatively, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, W138, etc. Additionally, host cells may be engineered to express various enzymes to ensure the proper processing of the collagen molecules. For example, the genes for prolyl 4-hydroxylase (i.e., the gene encoding the a subunit or prolyl 4-hydroxylase and the gene encoding the α subunit of prolyl 4-hydroxylase), may be coexpressed with the collagen gene in the host cell.

[0056] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express an α2 subunit of prolyl 4-hydroxylase of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with α2 subunit encoding DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express a desired α2 subunit of prolyl 4-hydroxylase.

[0057] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski (1962) Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al. (1980) Cell 22:817) genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al. (1980) Natl. Acad. Sci. USA 77:3567; O'Hare et al. (1981) Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al. (1981) J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al. (1984) Gene 30:147). Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047); and ODC (omithine decarboxylase) which confers resistance to the omithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L. (1987) In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory).

d. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS THAT EXPRESS THE α2 SUBUNIT PROTEIN OF THE INVENTION AND PURIFICATION OF THE EXPRESSED PROTEINS

[0058] The host cells which contain the coding sequence and which express the biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of “marker” gene functions; (c) assessing the level of transcription as measured by the expression of α2 subunit mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.

[0059] In the first approach, the presence of the enzyme coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the α2 subunit of prolyl 4-hydroxylase coding sequence, respectively, or portions or derivatives thereof.

[0060] In the second approach, the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the α2 subunit coding sequence is inserted within a marker gene sequence of the vector, recombinant cells containing coding sequence of the α2 subunit of prolyl 4-hydroxylase can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the α2 subunit sequence under the control of the same or different promoter used to control the expression of the α2 subunit coding sequence. Expression of the marker in response to induction or selection indicates expression of the α2 subunit coding sequence.

[0061] In the third approach, transcriptional activity of the α2 subunit coding region can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to the α2 subunit coding sequence or particular portions thereof. Alternatively, total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.

[0062] In the fourth approach, the expression of the enzyme product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.

[0063] The expressed enzyme of the invention, which is secreted into the culture medium, is purified to homogeneity, e.g., by chromatography. In one embodiment, the recombinant α2 subunit of prolyl 4-hydroxylase protein is purified by size exclusion chromatography.

[0064] However, other purification techniques known in the art can also be used, including ion exchange chromatography, and reverse-phase chromatography.

5. EXAMPLES

[0065] The invention will be further understood by reference to the following examples, which are intended to be purely exemplary of the invention.

Example 1 Isolation of Mouse CDNA Clones

[0066] A cDNA clone for the mouse α2 subunit, designated BT14.1, was obtained from a BALB/c mouse brain cDNA library in λgt10 (Clontech, Palo Alto Calif.) by using as a probe, a cDNA encoding the thymic shared antigen 1, as described in MacNeil, et al. (1993) J. Immunol. 151:6913-23. The BT14.1 clone had a high degree of homology to the human and chicken prolyl 4-hydroxylase a subunit. The cDNA clone BT14.1, however, did not contain sequences coding for the N-terminal region of the polypeptide. It was therefore used as a probe to screen mouse brain and skeletal muscle cDNA libraries.

[0067] Among 600,000 recombinants, 4 positive clones were obtained. Two of them, M1 and M4 were found to be identical, while M2 had a deletion and M3 contained two unrelated inserts. The clone M1, was used to screen 1.6×10⁶ plaques of a mouse skeletal muscle cDNA library in λgt10 (Clontech). One positive clone, M6, was obtained. This clone was characterized further and was found to be included in BT14.1. The 5′ ends of M1 and BT14.1 were at the same internal EcoRI site (at nucleotide position 220 of the sequence shown in FIG. 1). The extreme 5′ clone was isolated by using Ml to screen a mouse skeletal muscle cDNA library, and one positive clone was obtained, M6. As set forth below, at Example 2, the cDNA clones, considered in combination, cover the whole coding region of the mouse α2 subunit. cDNA clones for the mouse α1 subunit were then isolated by screening a 3T3 fibroblast λgt11 cDNA library (Clontech) with the human cDNA clone PA-49 for the α1 subunit, as described in Helaakoski et al. (1989) Proc. Natl. Acad. Sci. USA 86:4392-96, and eight positive clones were obtained out of 600,000 plaques.

[0068] Three of these clones, MA3, MA4, and MA7, were isolated and sequenced. The nucleotide and predicted amino acid sequences of the clones showed a significant similarity to those of the human and chick prolyl 4-hydroxylase a subunit. Two of the clones, MA3 and MA4, were found to represent the mouse counterparts of human mRNA containing the alternatively spliced exon 10 sequences, whereas MA7 contained exon 9 sequences. The cDNA clones did not contain the extreme 5′ end of the mRNA. Comparison of the cDNA derived amino acid sequences with those of the human and chick α1 subunits suggests that the cDNA clones cover the whole processed polypeptide but do not cover the 5′ untranslated region or the sequences corresponding to the N-terminal half of the signal peptide. See, GenBank database, accession no. U16162.

Example 2 Nucleotide Sequencing, Sequence Analysis, and Northern Blot Analysis

[0069] The nucleotide sequences for the clones described in Example 1 were determined by the dideoxynucleotide chain-termination method, as described in Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-67, with T7 DNA polymerase (Pharmacia, Peapack N.J.). Vector-specific or sequence-specific primers synthesized in an Applied Biosystems DNA synthesizer (Department of Biochemistry, University of Oulu) were used. The DNASIS and PROSIS version 6.00 sequence analysis software (Pharmacia), ANTHEPROT (as disclosed in Deleage et al. (1988) Comput. Appl. Biosci. 4:351-356), the Wisconsin Genetics Computer Group package version 8 (September 1994), and BOXSHADE (Kay Hofmann, Bioinformatics Group, Institut Suisse de Recherches Experimentales sur le Cancer Lausanne, Switzerland) were used to compile the sequence data.

[0070] The cDNA clones cover 2168 not of the corresponding mRNA and encode a 537-aa polypeptide (FIG. 1). A putative signal peptide is present at the N terminus of the deduced polypeptide, the most likely first amino acid of the mature α2 subunit being tryptophan, based on the computational parameters of von Hejne (1986) Nucleic Acid Res. 14:4683-90, which means that the size of the signal sequence would be 19 aa and that of the processed α2 subunit 518 aa. The molecular weight of the processed polypeptide is 59,000. The cDNA clones also cover 150 bp and 407 bp of the 5′ and 3′ untranslated sequences, respectively (FIG. 1). The 3′ untranslated sequence contains a canonical polyadenylylation signal, which is accompanied 12 nucleotides downstream by a poly(A) tail of 15 nucleotide position.

[0071] The mouse α2 and mouse α1 polypeptides are of similar sizes, α2 being 518 and α1 517 amino acids, assuming that the α2 polypeptide begins with a tryptophan residue and α1 with a histidine residue, as does the human α1 polypeptide. The processed human α1 subunit contains 517 amino acids and the chick α1 subunit 516 amino acids (as described in Bassuk, et al., supra), whereas the processed C. elegans a subunit is longer, 542 aa (Veijola, et al., supra), the difference being mainly due to a 32 aa extension present in the C terminus of the polypeptide (FIG. 2).

[0072] The mouse α2 and α1 subunits contain two potential attachment sites for asparagine-linked oligosaccharides; the positions of the -Asn-Leu-Ser-and -Asn-Glu-Thr- sequences of the α2 subunit are indicated in FIG. 1. The positions of the five cysteine residues present in the human, mouse, and chicken α1 subunits and the C. elegans a subunit are all conserved in the α2 subunit, but the latter contains an additional cysteine between the fourth and fifth cysteines of the α1 subunits. Interestingly, this is located at a site where the conserved stretch of amino acids is also interrupted in the mouse α1 and C. elegans a subunits.

[0073] The overall amino acid sequence identity and similarity between the mouse α2 and mouse al subunits are 63% and 83%, respectively, and those between the mouse α2 and C. elegans a subunits are 41% and 67%, respectively, which are almost the same as between the mouse α1 and C. elegans a subunits, 43% and 67%. The identity is not distributed equally, however, being highest within the C-terminal domain, which is believed to represent the catalytically important part of the α1 subunit (id.; Myllyla et al. (1992) Biochem. J. 286:923-927). The two histidines, residues 412 and 483 in the mouse α1 subunit (FIG. 2), that have been suggested to be involved in the Fe²⁺ binding sites of prolyl 4-hydroxylase are both conserved and are both located within the conserved C-terminal domain.

[0074] A mouse multitissue Northern blot (Clontech) containing 2 μg of poly(A)′ RNA per sample isolated from various mouse tissues was hybridized under the stringent conditions suggested in the manufacture's instructions. The probe used was ³²P labeled cDNA clone BT14.1 or MA7.

[0075] The expression patterns of both types of a α-subunit mRNA were found to be very similar, the intensities of the hybridization signals being highest in the heart, lung, and brain. The size of the α2 subunit mRNA was 2.4 kb. The mouse α1 subunit was found to have two mRNA transcripts, at least in the heart, brain, and lungs: the more intense the signal was at 3.4 kb and the weaker one at 4.3 kb.

Example 3 Cell Cultures and Generation of Recombinant Baculoviruses

[0076] Since it was not known initially whether the α2 polypeptide represented an a subunit of prolyl 4-hydroxylase, a subunit of prolyl 3-hydroxylase, or some other 2-oxoglutarate dioxygenase, a recombinant polypeptide was expressed in insect cells to elucidate its function. Specifically, Spodopiera frugiperda Sf9 insect cells were cultured at 27° C. in TNM-FH medium (Sigma-Aldrich, St. Louis Mo.) supplemented with 10% fetal bovine serum (Invitrogen). To construct an α(11)-subunit cDNA for expression, the clone BT14.1 was digested with the BamHI and EcoRI restriction enzymes, giving a fragment encompassing bp 592-2168. The 5′ fragment was amplified from the X DNA of M6. The primers used were cDNA specific, M3PH (5′-AAGTTGCGGCCGCGAGCATCAGCAAGGTACTGC-3′) (SEQ ID NO: 19), containing an artificial NotI site and M65′PCR (5′-TCTCCGGATCCAGTTTGTACGTGTC-3′) (SEQ ID NO:20), containing a natural BamHI site. PCR was performed under the conditions recommended by the supplier of the Taq polymerase (Promega, Madison Wis.), and the reactions were cycled 27 times as follows: denaturation at 94° C. for 1 min, annealing at 66° C. for 1 min, and extension at 72° C. for 3 min. The product was digested with Not I and BamHI restriction enzymes to give a fragment that extended from bp 120 to 591. The two Not I-BamHI and BamNI-EcoRI fragments were then cloned into the PBLUESCRIPT vector (Stratagene, La Jolla Calif.), the construct was digested with Not I and EcoRV, and the resulting fragment was ligated into a Not I-Sma I site of the baculovirus transfer vector pVL1392, wherein said vector was obtained according to the methods described in Luckow and Summers (1989) Virology 170:31-39. The pVI construct was cotransfected into Sf9 insect cells with a modified Autographa californica nuclear polyhedrosis virus DNA by using the BACULOGOLD transfection kit (PharMingen, San Diego Calif.). The resultant viral pool was collected 4 days later, amplified, and plague purified. The recombinant virus was checked by PCR-based methods, as described in Malitschek and Schartl (1991) BioTechniques 11:177-178.

Example 4 Expression and Analysis of Recombinant Proteins

[0077] A recombinant baculovirus coding for the mouse α2 subunit was generated and used to infect S. frugiperda insect cells with or without the human PDI/β subunit, wherein the insect cells were infected at a multiplicity of 5. For production of an enzyme tetramer, the human α59 1 (see, Vuori, et al., supra) or mouse α2 viruses and the PDI/β viruses (id.) were used in a 1:1 or 2:1 ratio. The cells were harvested 72 hours after infection, homogenized in 0.01 M tris, pH 7.8/0.1 M NaCl/0.1 M glycine/10 μM dithiothreitol/0.1% Triton X-100, and centrifuged. The resulting supematants were analyzed by SDS/8% PAGE or nondenaturing 7.5% PAGE and assayed for enzyme activities. The cell pellets were further solubilized in 1% SDS, and the 0.1% Triton X-100-soluble and 1% SDS-soluble proteins were analyzed by SDS/PAGE under reducing for the α1 subunit of prolyl 4-hydroxylase (Veijola et al., supra; Vuori et al., supra; John et al. (1993) EMBO J. 2:1587-95). The polypeptide formed insoluble aggregates, and efficient extraction of the recombinant mouse α2 subunit from the cell homogenates required the use of 1% SDS.

Example 5 Enzyme Activity Assays

[0078] Prolyl 4-hydroxylase activity was assayed by a method based on the decarboxylation of 2-oxoH ¹⁴C-glutarate, as disclosed in Kivirriko and Myllyla (1982) Methods Enzymol. 82:245-304. The K_(m) values were determined by varying the concentration of one substrate in the presence of fixed concentrations of the second while the concentrations of the other substrates were kept constant, as set forth in Myllyla et al. (1977) Eur. J. Biochem. 80:349-357.

[0079] The 0.1% Triton X-100 extracts from cell homogenates containing either the mouse-human type II or the human type I enzyme were analyzed for prolyl 4-hydroxylase activity with an assay based on the hydroxylation-coupled decarbosylation of 2-oxo[1¹⁴C]glutarate (Kivirikko and Myllyla, supra). The activities were very similar for both.

[0080] To show that the activity of the mouse/human type 2 enzyme was prolyl 4-hydroxylase activity, the amount of 4-hydroxyproline in a (Pro-Pro-Gly)₁₀ substrate was determined after the reaction. The values indicated that the type 2 and type 1 enzymes behaved very similarly and that the activity of the type 2 enzyme was indeed prolyl 4-hydroxylase activity. The K_(m) values for Fe²⁺, 2-oxoglutarate, and ascorbate and the K_(i) value for pyridine-2,4,-dicarboxylate, which acts as a competitive inhibitor with respect to 2-oxoglutarate, were likewise highly similar for the two enzymes, as shown in Table I. TABLE I K_(m) values for cosubstrates and the peptide substrate and K₁ values for certain inhibitors of the human type 1 and mouse/human type 2 prolyl 4-hydroxylase tetramers. K_(m) or K_(i), μM Cosubstrate, substrate, or inhibitor Constant α1₂β₂ α2₂β₂ Fe²⁺ K_(m) 4  4 2-Oxoglutatrate K_(m) 22 12 Ascorbate K_(m) 330 340  (Pro--Pro--Gly) K_(m) 18 45 Poly(t-proline), M_(t) 7000 K_(i) 0.5 300* Poly(t-proline), M_(t) 44,000 K_(i) 0.02  30* Pyridine-2,4-dicarboxylate K_(i) 2  1

[0081] Notably, the values differed distinctly in that the type 2 enzyme was inhibited by poly (L-proline) only at very high concentrations. As poly (L-proline) is a well-recognized, effective competitive inhibitor of type 1 prolyl 4-hydroxylase from all vertebrate sources studied and as poly (L-proline) is an effective polypeptide substrate for all plant prolyl 4-hydroxylases studied. Such finding was unexpected. Distinct differences thus appear to exist in the structures of the peptide binding sites of various prolyl 4-hydroxylases, but no detailed data are currently available on this aspect.

Example 6 Expression of the Mouse α2 Subunit and an Active Mouse α2 PDI/βEnzyme Tetramer in Insect Cells

[0082] Insect cells were coinfected with two recombinant viruses coding for the two polypeptides in order to study whether an association between the mouse α2 subunit and the human PDI/β-subunit could be achieved. A hybrid protein was formed and was soluble in a buffer containing 0.1% Triton X-100, as shown by PAGE performed under nondenaturing conditions. The mouse α2 subunit expressed alone did not give any extractable recombinant protein under the same conditions, termed here the type 1 tetramer, indicating that the hybrid protein is likely to be an α2₂β₂ tetramer, termed the type 2 tetramer. No difference was found in the association of the α2 and α1 subunits with the PDI/β subunit into the tetramer. To show that the hybrid protein formed contains the human PDI/β subunit, Western blotting was performed. When the mouse α2 subunit was expressed together with the human PDI/β subunit, the protein complex contained the PDI/β subunit.

Example 7 Isolation and Sequencing of Human α2 Subunit Gene

[0083] A human lung fibroblast genomic library (cloned in the lamda FIX vector (Stratagene)) and a human chromosome 5 library (cloned in the lamda vector Charon 40 (American Type Culture Collection, Manassas Va.)) were screened with probes comprising ³²P-labelled nick-translated PCR fragments corresponding to the previously characterized human prolyl 4-hydroxylase a subunit cDNA sequence.

[0084] Positive clones from both the human lung fibroblast library and the human chromosome 5 library were identified, isolated and analyzed by southern blotting. Suitable fragments were subcloned into pSP72 vector (Promega) for further analysis.

[0085] Five positive clones, designated GL-2, GL-5, GL-20, GL-141 and GL-142 were obtained from the human lung fibroblast genomic library. Two of these clones, GL-2 and GL-141 were identical. Clones corresponding to the 5′ and 3′-ends of the gene encoding the α2 subunit of prolyl 4-hydroxylase were not obtained.

[0086] The human chromosome 5 library was screened twice with two separate probes. The first probe corresponded to the 5′-end of the previously characterized cDNA sequence for α2 subunit of prolyl 4-hydroxylase. The second probe corresponded to the 3′-end of the same cDNA sequence. Several positive clones were obtained, including GL-3, GL-4, GL-9, GL-11, GL-11B, and GL-156GL-3, GL-4, GL-9 and GL-11B corresponded to the 5′-end of the protein. GL-11A and GL-156 corresponded to the 3′-end of the protein clones GL-11A and GL-156 were found to be identical.

[0087] The derived sequence corresponding to the gene is more than 30 kb in size and is comprised of 15 exons. The exons that encode solely protein sequences vary from 54 to 240 base pairs and the introns vary from 241 to at least 3200 base pairs (see, FIGS. 2-9).

[0088] As compared to the gene sequence for the α1 subunit, only one exon of the α2 subunit corresponds to the two mutually exclusive spliced exons of the a l subunit gene (EXON 9 of the α1 subunit gene).

[0089] The deduced amino acid sequence is 63% homologous to the known α(1) subunit.

Example 8 Expression of the Human α2 Subunit of Prolyl 4-Hydroxylase in Insect Cells

[0090] Using the methods of Examples 3, 4 and 6, the α2 subunit isoform of prolyl 4-hydroxylase was expressed and analyzed. Expression data in insect cells demonstrated that the α2 subunit isoform forms an active type 2 prolyl-4-hydroxyl α₂β₂ tetramer with the human β subunit.

[0091] Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. It is also to be understood that all base pair sizes given for nucleotides are approximate and are used for purposes of description.

[0092] All references cited herein are hereby incorporated by reference in their entirety.

1 31 2168 base pairs nucleic acid unknown unknown cDNA CDS 151..1761 1 GCAGTTTCAG AGACCGGTGG CGATTGGCTG ACTGATTCAA CAAATAGAGC ATTCTCTGTG 60 CCTGGAGACT TTCAAGGACT GAGGCAGGCA GAAGGGAAGA CTCAGAAAGT TCAGGTCCAG 120 AGCATCAGCA AGGTACTGCC CTTTCCAGTT ATG AAG CTC CAG GTG TTG GTG TTG 174 Met Lys Leu Gln Val Leu Val Leu 1 5 GTG TTG CTG ATG TCC TGG TTC GGT GTC CTG AGC TGG GTG CAG GCA GAA 222 Val Leu Leu Met Ser Trp Phe Gly Val Leu Ser Trp Val Gln Ala Glu 10 15 20 TTC TTC ACC TCC ATT GGG CAC ATG ACC GAT CTG ATT TAC GCA GAG AAG 270 Phe Phe Thr Ser Ile Gly His Met Thr Asp Leu Ile Tyr Ala Glu Lys 25 30 35 40 GAC CTG GTA CAG TCT CTG AAG GAG TAC ATC CTT GTG GAG GAA GCC AAG 318 Asp Leu Val Gln Ser Leu Lys Glu Tyr Ile Leu Val Glu Glu Ala Lys 45 50 55 CTC GCC AAG ATT AAG AGC TGG GCC AGC AAG ATG GAA GCC CTG ACC AGC 366 Leu Ala Lys Ile Lys Ser Trp Ala Ser Lys Met Glu Ala Leu Thr Ser 60 65 70 AGA TCA GCT GCC GAC CCC GAG GGC TAC CTG GCT CAT CCT GTG AAT GCC 414 Arg Ser Ala Ala Asp Pro Glu Gly Tyr Leu Ala His Pro Val Asn Ala 75 80 85 TAC AAG CTG GTG AAG CGG TTG AAC ACA GAC TGG CCT GCA CTG GGG GAC 462 Tyr Lys Leu Val Lys Arg Leu Asn Thr Asp Trp Pro Ala Leu Gly Asp 90 95 100 CTT GTC CTT CAG GAT GCT TCG GCA GGT TTT GTC GCT AAC CTC TCA GTT 510 Leu Val Leu Gln Asp Ala Ser Ala Gly Phe Val Ala Asn Leu Ser Val 105 110 115 120 CAG CGG CAA TTC TTC CCC ACT GAT GAG GAC GAG TCT GGA GCT GCC AGA 558 Gln Arg Gln Phe Phe Pro Thr Asp Glu Asp Glu Ser Gly Ala Ala Arg 125 130 135 GCC CTG ATG AGA CTT CAG GAC ACG TAC AAA CTG GAT CCG GAC ACG ATT 606 Ala Leu Met Arg Leu Gln Asp Thr Tyr Lys Leu Asp Pro Asp Thr Ile 140 145 150 TCC AGA GGG GAA CTT CCA GGC ACA AAG TAC CAG GCC ATG CTG AGT GTG 654 Ser Arg Gly Glu Leu Pro Gly Thr Lys Tyr Gln Ala Met Leu Ser Val 155 160 165 GAC GAC TGC TTT GGG CTG GGC CGC TCA GCT TAC AAT GAA GGA GAC TAT 702 Asp Asp Cys Phe Gly Leu Gly Arg Ser Ala Tyr Asn Glu Gly Asp Tyr 170 175 180 TAC CAT ACT GTG CTG TGG ATG GAG CAG GTA CTG AAG CAG CTC GAT GCT 750 Tyr His Thr Val Leu Trp Met Glu Gln Val Leu Lys Gln Leu Asp Ala 185 190 195 200 GGG GAG GAG GCC ACT GTT ACC AAG TCC CTG GTG CTG GAC TAC CTG AGC 798 Gly Glu Glu Ala Thr Val Thr Lys Ser Leu Val Leu Asp Tyr Leu Ser 205 210 215 TAT GCT GTC TTC CAA CTG GGT GAC CTG CAC CGT GCT GTG GAA CTC ACC 846 Tyr Ala Val Phe Gln Leu Gly Asp Leu His Arg Ala Val Glu Leu Thr 220 225 230 CGC CGC CTG CTC TCT CTT GAC CCA AGC CAC GAA CGA GCT GGA GGG AAT 894 Arg Arg Leu Leu Ser Leu Asp Pro Ser His Glu Arg Ala Gly Gly Asn 235 240 245 CTG CGG TAC TTT GAA CGG TTG TTA GAG GAA GAA AGA GGG AAA TCA CTG 942 Leu Arg Tyr Phe Glu Arg Leu Leu Glu Glu Glu Arg Gly Lys Ser Leu 250 255 260 TCA AAT CAG ACA GAC GCC GGA CTG GCC ACC CAG GAA AAC TTG TAC GAG 990 Ser Asn Gln Thr Asp Ala Gly Leu Ala Thr Gln Glu Asn Leu Tyr Glu 265 270 275 280 AGG CCC ACG GAC TAC CTG CCT GAG AGG GAT GTG TAC GAG AGC CTG TGT 1038 Arg Pro Thr Asp Tyr Leu Pro Glu Arg Asp Val Tyr Glu Ser Leu Cys 285 290 295 CGA GGG GAG GGC GTG AAA CTG ACA CCC CGG AGG CAG AAG AAG CTT TTC 1086 Arg Gly Glu Gly Val Lys Leu Thr Pro Arg Arg Gln Lys Lys Leu Phe 300 305 310 TGT AGG TAC CAT CAT GGA AAC AGA GTG CCA CAG CTC CTC ATC GCC CCC 1134 Cys Arg Tyr His His Gly Asn Arg Val Pro Gln Leu Leu Ile Ala Pro 315 320 325 TTC AAA GAG GAA GAC GAG TGG GAC AGC CCA CAC ATC GTC AGG TAC TAT 1182 Phe Lys Glu Glu Asp Glu Trp Asp Ser Pro His Ile Val Arg Tyr Tyr 330 335 340 GAT GTG ATG TCC GAC GAA GAA ATC GAG AGG ATC AAG GAG ATT GCT AAG 1230 Asp Val Met Ser Asp Glu Glu Ile Glu Arg Ile Lys Glu Ile Ala Lys 345 350 355 360 CCC AAA CTT GCA CGA GCC ACT GTG CGT GAC CCC AAG ACA GGT GTC CTC 1278 Pro Lys Leu Ala Arg Ala Thr Val Arg Asp Pro Lys Thr Gly Val Leu 365 370 375 ACT GTT GCC AGC TAC AGA GTT TCC AAA AGC TCC TGG CTA GAG GAG GAT 1326 Thr Val Ala Ser Tyr Arg Val Ser Lys Ser Ser Trp Leu Glu Glu Asp 380 385 390 GAC GAC CCT GTT GTG GCC CGG GTC AAC CGG CGG ATG CAA CAT ATC ACC 1374 Asp Asp Pro Val Val Ala Arg Val Asn Arg Arg Met Gln His Ile Thr 395 400 405 GGG CTA ACG GTG AAG ACT GCA GAG CTA TTG CAG GTC GCA AAC TAC GGA 1422 Gly Leu Thr Val Lys Thr Ala Glu Leu Leu Gln Val Ala Asn Tyr Gly 410 415 420 ATG GGG GGA CAG TAC GAA CCA CAC TTT GAC TTC TCA AGG AGC GAT GAC 1470 Met Gly Gly Gln Tyr Glu Pro His Phe Asp Phe Ser Arg Ser Asp Asp 425 430 435 440 GAA GAT GCT TTC AAG CGT TTA GGG ACT GGG AAC CGT GTG GCC ACG TTT 1518 Glu Asp Ala Phe Lys Arg Leu Gly Thr Gly Asn Arg Val Ala Thr Phe 445 450 455 CTA AAC TAC ATG AGC GAT GTC GAA GCT GGT GGT GCC ACC GTC TTT CCT 1566 Leu Asn Tyr Met Ser Asp Val Glu Ala Gly Gly Ala Thr Val Phe Pro 460 465 470 GAC TTG GGA GCT GCT ATT TGG CCC AAG AAG GGC ACA GCT GTA TTC TGG 1614 Asp Leu Gly Ala Ala Ile Trp Pro Lys Lys Gly Thr Ala Val Phe Trp 475 480 485 TAC AAC CTT CTT CGC AGT GGG GAA GGT GAT TAT CGG ACG AGA CAT GCA 1662 Tyr Asn Leu Leu Arg Ser Gly Glu Gly Asp Tyr Arg Thr Arg His Ala 490 495 500 GCC TGC CCT GTG CTT GTG GGC TGC AAG TGG GTC TCC AAC AAG TGG TTC 1710 Ala Cys Pro Val Leu Val Gly Cys Lys Trp Val Ser Asn Lys Trp Phe 505 510 515 520 CAT GAG CGA GGA CAG GAG TTC TTA AGA CCT TGT GGA ACA ACG GAA GTT 1758 His Glu Arg Gly Gln Glu Phe Leu Arg Pro Cys Gly Thr Thr Glu Val 525 530 535 GAT TGACGTCCTT TTCTCTCCGC TCCTCCCTGG CCCATAGTCC AAATCATCTT 1811 Asp CAAGTTCAAC ATGACAGCTT CCTTTTTTAT GTCCCAGCTC CTGTCAGGCA GGTCATTGGA 1871 GGAGCCAGTG TTTGACTGAA TTGAGAGAGT ATATCCTGAG CCTAGTCCTG GGTGACCTGG 1931 GCCCCAGACT CTGACCAGCT TACACCTGCC CTGGCTCTGG GGGTGTCTTG GCATGGCTGC 1991 GGTAGAGCCA GACTATAGCA CCCGGCACGG TCGCTTTGTA CCTCAGATAT TTCAGGTAGA 2051 AGATGTCTCA GTGAAACCAA AGTTCTGATG CTGTTTACAT GTGTGTTTTT ATCACATTTC 2111 TATTTGTTGT GGCTTTAACC AAAAAATAAA AATGTTCCTG CCAAAAAAAA AAAAAAA 2168 537 amino acids amino acid linear protein 2 Met Lys Leu Gln Val Leu Val Leu Val Leu Leu Met Ser Trp Phe Gly 1 5 10 15 Val Leu Ser Trp Val Gln Ala Glu Phe Phe Thr Ser Ile Gly His Met 20 25 30 Thr Asp Leu Ile Tyr Ala Glu Lys Asp Leu Val Gln Ser Leu Lys Glu 35 40 45 Tyr Ile Leu Val Glu Glu Ala Lys Leu Ala Lys Ile Lys Ser Trp Ala 50 55 60 Ser Lys Met Glu Ala Leu Thr Ser Arg Ser Ala Ala Asp Pro Glu Gly 65 70 75 80 Tyr Leu Ala His Pro Val Asn Ala Tyr Lys Leu Val Lys Arg Leu Asn 85 90 95 Thr Asp Trp Pro Ala Leu Gly Asp Leu Val Leu Gln Asp Ala Ser Ala 100 105 110 Gly Phe Val Ala Asn Leu Ser Val Gln Arg Gln Phe Phe Pro Thr Asp 115 120 125 Glu Asp Glu Ser Gly Ala Ala Arg Ala Leu Met Arg Leu Gln Asp Thr 130 135 140 Tyr Lys Leu Asp Pro Asp Thr Ile Ser Arg Gly Glu Leu Pro Gly Thr 145 150 155 160 Lys Tyr Gln Ala Met Leu Ser Val Asp Asp Cys Phe Gly Leu Gly Arg 165 170 175 Ser Ala Tyr Asn Glu Gly Asp Tyr Tyr His Thr Val Leu Trp Met Glu 180 185 190 Gln Val Leu Lys Gln Leu Asp Ala Gly Glu Glu Ala Thr Val Thr Lys 195 200 205 Ser Leu Val Leu Asp Tyr Leu Ser Tyr Ala Val Phe Gln Leu Gly Asp 210 215 220 Leu His Arg Ala Val Glu Leu Thr Arg Arg Leu Leu Ser Leu Asp Pro 225 230 235 240 Ser His Glu Arg Ala Gly Gly Asn Leu Arg Tyr Phe Glu Arg Leu Leu 245 250 255 Glu Glu Glu Arg Gly Lys Ser Leu Ser Asn Gln Thr Asp Ala Gly Leu 260 265 270 Ala Thr Gln Glu Asn Leu Tyr Glu Arg Pro Thr Asp Tyr Leu Pro Glu 275 280 285 Arg Asp Val Tyr Glu Ser Leu Cys Arg Gly Glu Gly Val Lys Leu Thr 290 295 300 Pro Arg Arg Gln Lys Lys Leu Phe Cys Arg Tyr His His Gly Asn Arg 305 310 315 320 Val Pro Gln Leu Leu Ile Ala Pro Phe Lys Glu Glu Asp Glu Trp Asp 325 330 335 Ser Pro His Ile Val Arg Tyr Tyr Asp Val Met Ser Asp Glu Glu Ile 340 345 350 Glu Arg Ile Lys Glu Ile Ala Lys Pro Lys Leu Ala Arg Ala Thr Val 355 360 365 Arg Asp Pro Lys Thr Gly Val Leu Thr Val Ala Ser Tyr Arg Val Ser 370 375 380 Lys Ser Ser Trp Leu Glu Glu Asp Asp Asp Pro Val Val Ala Arg Val 385 390 395 400 Asn Arg Arg Met Gln His Ile Thr Gly Leu Thr Val Lys Thr Ala Glu 405 410 415 Leu Leu Gln Val Ala Asn Tyr Gly Met Gly Gly Gln Tyr Glu Pro His 420 425 430 Phe Asp Phe Ser Arg Ser Asp Asp Glu Asp Ala Phe Lys Arg Leu Gly 435 440 445 Thr Gly Asn Arg Val Ala Thr Phe Leu Asn Tyr Met Ser Asp Val Glu 450 455 460 Ala Gly Gly Ala Thr Val Phe Pro Asp Leu Gly Ala Ala Ile Trp Pro 465 470 475 480 Lys Lys Gly Thr Ala Val Phe Trp Tyr Asn Leu Leu Arg Ser Gly Glu 485 490 495 Gly Asp Tyr Arg Thr Arg His Ala Ala Cys Pro Val Leu Val Gly Cys 500 505 510 Lys Trp Val Ser Asn Lys Trp Phe His Glu Arg Gly Gln Glu Phe Leu 515 520 525 Arg Pro Cys Gly Thr Thr Glu Val Asp 530 535 2207 base pairs nucleic acid unknown unknown cDNA CDS 1..2205 3 GGG GAA GGA ACA CTG TAG GGG ATA GCT GTC CAC GGA CGC TGT CTA CAA 48 Gly Glu Gly Thr Leu * Gly Ile Ala Val His Gly Arg Cys Leu Gln 540 545 550 GAC CCT GGA GTG AGA TAA CGT GCC TGG TAC TGT GCC CTG CAT GTG TAA 96 Asp Pro Gly Val Arg * Arg Ala Trp Tyr Cys Ala Leu His Val * 555 560 565 GAT GCC CAG TTG ACC TTC GCA GCA GGA GCC TGG ATC AGG CAC TTC CTG 144 Asp Ala Gln Leu Thr Phe Ala Ala Gly Ala Trp Ile Arg His Phe Leu 570 575 580 585 CCT CAG GTA TTG CTG GAC AGC CCA GAC ACT TCC CTC TGT GAC CAT GAA 192 Pro Gln Val Leu Leu Asp Ser Pro Asp Thr Ser Leu Cys Asp His Glu 590 595 600 ACT CTG GGT GTC TGC ATT GCT GAT GGC CTG GTT TGG TGT CCT GAG CTG 240 Thr Leu Gly Val Cys Ile Ala Asp Gly Leu Val Trp Cys Pro Glu Leu 605 610 615 TGT GCA GGC CGA ATT CTT CAC CTC TAT TGG GCA CAT GAC TGA CCT GAT 288 Cys Ala Gly Arg Ile Leu His Leu Tyr Trp Ala His Asp * Pro Asp 620 625 630 TTA TGC AGA GAA AGA GCT GGT GCA GTC TCT GAA AGA GTA CAT CCT TGT 336 Leu Cys Arg Glu Arg Ala Gly Ala Val Ser Glu Arg Val His Pro Cys 635 640 645 GGA GGA AGC CAA GCT TTC CAA GAT TAA GAG CTG GGC CAA CAA AAT GGA 384 Gly Gly Ser Gln Ala Phe Gln Asp * Glu Leu Gly Gln Gln Asn Gly 650 655 660 665 AGC CTT GAC TAG CAA GTC AGC TGC TGA TGC TGA GGG CTA CCT GGC TCA 432 Ser Leu Asp * Gln Val Ser Cys * Cys * Gly Leu Pro Gly Ser 670 675 680 CCC TGT GAA TGC CTA CAA ACT GGT GAA GCG GCT AAA CAC AGA CTG GCC 480 Pro Cys Glu Cys Leu Gln Thr Gly Glu Ala Ala Lys His Arg Leu Ala 685 690 695 TGC GCT GGA GGA CCT TGT CCT GCA GGA CTC AGC TGC AGG TTT TAT CGC 528 Cys Ala Gly Gly Pro Cys Pro Ala Gly Leu Ser Cys Arg Phe Tyr Arg 700 705 710 CAA CCT CTC TGT GCA GCG GCA GTT CTT CCC CAC TGA TGA GGA CGA GAT 576 Gln Pro Leu Cys Ala Ala Ala Val Leu Pro His * * Gly Arg Asp 715 720 725 AGG AGC TGC CAA AGC CCT GAT GAG ACT TCA GGA CAC ATA CAG GCT GGA 624 Arg Ser Cys Gln Ser Pro Asp Glu Thr Ser Gly His Ile Gln Ala Gly 730 735 740 745 CCC AGG CAC AAT TTC CAG AGG GGA ACT TCC AGG AAC CAA GTA CCA GGC 672 Pro Arg His Asn Phe Gln Arg Gly Thr Ser Arg Asn Gln Val Pro Gly 750 755 760 AAT GCT GAG TGT GGA TGA CTG CTT TGG GAT GGG CCG CTC GGC CTA CAA 720 Asn Ala Glu Cys Gly * Leu Leu Trp Asp Gly Pro Leu Gly Leu Gln 765 770 775 TGA AGG GGA CTA TTA TCA TAC GGT GTT GTG GAT GGA GCA GGT GCT AAA 768 * Arg Gly Leu Leu Ser Tyr Gly Val Val Asp Gly Ala Gly Ala Lys 780 785 790 GCA GCT TGA TGC CGG GGA GGA GGC CAC CAC AAC CAA GTC ACA GGT GCT 816 Ala Ala * Cys Arg Gly Gly Gly His His Asn Gln Val Thr Gly Ala 795 800 805 GGA CTA CCT ACG CTA TGC TGT CTT CCA GTT GGG TGA TCT GCA CCG TGC 864 Gly Leu Pro Thr Leu Cys Cys Leu Pro Val Gly * Ser Ala Pro Cys 810 815 820 825 CCT GGA GCT CAC CCG CCG CCT GCT CTC CCT TGA CCC AAG CCA CGA ACG 912 Pro Gly Ala His Pro Pro Pro Ala Leu Pro * Pro Lys Pro Arg Thr 830 835 840 AGC TGG AGG GAA TCT GCG GTA CTT TGA GCA GTT ATT GGA GGA AGA GAG 960 Ser Trp Arg Glu Ser Ala Val Leu * Ala Val Ile Gly Gly Arg Glu 845 850 855 AGA AAA AAC GTT AAC AAA TCA GAC AGA AGC TGA GCT AGC AAC CCC AGA 1008 Arg Lys Asn Val Asn Lys Ser Asp Arg Ser * Ala Ser Asn Pro Arg 860 865 870 AGG CAT CTA TGA GAG GCC TGT GGA CTA CCT GCC TGA GAG GGA TGT TTA 1056 Arg His Leu * Glu Ala Cys Gly Leu Pro Ala * Glu Gly Cys Leu 875 880 885 CGA GAG CCT CTG TCG TGG GGA GGG TGT CAA ACC TCG CAA GCA TAG ACA 1104 Arg Glu Pro Leu Ser Trp Gly Gly Cys Gln Thr Ser Gln Ala * Thr 890 895 900 905 GAA GAG GCT TTT CTG TAG GTA CCA CCA TGG CAA CAG GGC CCC ACA GCT 1152 Glu Glu Ala Phe Leu * Val Pro Pro Trp Gln Gln Gly Pro Thr Ala 910 915 920 GCT CAT TGC CCC CTT CAA AGA GGA GGA CGA GTG GGA CAG CCC GCA CAT 1200 Ala His Cys Pro Leu Gln Arg Gly Gly Arg Val Gly Gln Pro Ala His 925 930 935 CGT CAG GTA CTA CGA TGT CAT GTC TGA TGA GGA AAT CGA GAG GAT CAA 1248 Arg Gln Val Leu Arg Cys His Val * * Gly Asn Arg Glu Asp Gln 940 945 950 GGA GAT CGC AAA ACC TAA ACT TGC ACG AGC CAC CGT TCG TGA TCC CAA 1296 Gly Asp Arg Lys Thr * Thr Cys Thr Ser His Arg Ser * Ser Gln 955 960 965 GAG AGG AGT CCT CAC TGT CGC CAG CTA CCG GGT TTC CAA AAG CTC CTG 1344 Glu Arg Ser Pro His Cys Arg Gln Leu Pro Gly Phe Gln Lys Leu Leu 970 975 980 985 GCT AGA GGA AGA TGA TGA CCC TGT TGT GGC CCG AGT AAA TCG TCG GAT 1392 Ala Arg Gly Arg * * Pro Cys Cys Gly Pro Ser Lys Ser Ser Asp 990 995 1000 GCA GCA TAT CAC AGG GTT AAC AGT AAA GAC TGC AGA ATT GTT ACA GGT 1440 Ala Ala Tyr His Arg Val Asn Ser Lys Asp Cys Arg Ile Val Thr Gly 1005 1010 1015 TGC AAA TTA TGG AGT GGG AGG ACA GTA TGA ACC GCA CTT CGA CTT CTC 1488 Cys Lys Leu Trp Ser Gly Arg Thr Val * Thr Ala Leu Arg Leu Leu 1020 1025 1030 TAG GAA TGA TGA GCG AGA TAC TTT CAA GCA TTT AGG GAC GGG GAA TCG 1536 * Glu * * Ala Arg Tyr Phe Gln Ala Phe Arg Asp Gly Glu Ser 1035 1040 1045 TGT GGC TAC TTT CTT AAA CTA CAT GAG TGA TGT AGA AGC TGG TGG TGC 1584 Cys Gly Tyr Phe Leu Lys Leu His Glu * Cys Arg Ser Trp Trp Cys 1050 1055 1060 1065 CAC CGT CTT CCC TGA TCT GGG GGC TGC AAT TTG GCC TAA GAA GGG TAC 1632 His Arg Leu Pro * Ser Gly Gly Cys Asn Leu Ala * Glu Gly Tyr 1070 1075 1080 AGC TGT GTT CTG GTA CAA CCT CTT GCG GAG CGG GGA AGG TGA CTA CCG 1680 Ser Cys Val Leu Val Gln Pro Leu Ala Glu Arg Gly Arg * Leu Pro 1085 1090 1095 AAC AAG ACA TGC TGC CTG CCC TGT GCT TGT GGG CTG CAA GTG GGT CTC 1728 Asn Lys Thr Cys Cys Leu Pro Cys Ala Cys Gly Leu Gln Val Gly Leu 1100 1105 1110 CAA TAA GTG GTT CCA TGA ACG AGG ACA GGA GTT CTT GAG ACC TTG TGG 1776 Gln * Val Val Pro * Thr Arg Thr Gly Val Leu Glu Thr Leu Trp 1115 1120 1125 ATC AAC AGA AGT TGA CTG ACA TCC TTT TCT GTC CTT CCC CTT CCT GGT 1824 Ile Asn Arg Ser * Leu Thr Ser Phe Ser Val Leu Pro Leu Pro Gly 1130 1135 1140 1145 CCT TCA GCC CAT GTC AAC GTG ACA GAC ACC TTT GTA TGT TCC TTT GTA 1872 Pro Ser Ala His Val Asn Val Thr Asp Thr Phe Val Cys Ser Phe Val 1150 1155 1160 TGT TCC TAT CAG GCT GAT TTT TGG AGA AAT GAA TGT TTG TCT GGA GCA 1920 Cys Ser Tyr Gln Ala Asp Phe Trp Arg Asn Glu Cys Leu Ser Gly Ala 1165 1170 1175 GAG GGA GAC CAT ACT AGG GCG ACT CCT GTG TGA CTG AAG TCC CAG CCC 1968 Glu Gly Asp His Thr Arg Ala Thr Pro Val * Leu Lys Ser Gln Pro 1180 1185 1190 TTC CAT TCA GCC TGT GCC ATC CCT GGC CCC AAG GCT AGG ATC AAA GTG 2016 Phe His Ser Ala Cys Ala Ile Pro Gly Pro Lys Ala Arg Ile Lys Val 1195 1200 1205 GCT GCA GCA GAG TTA GCT TGC TAG CGC CTA GCA AGG TGC CTT TGT ACC 2064 Ala Ala Ala Glu Leu Ala Cys * Arg Leu Ala Arg Cys Leu Cys Thr 1210 1215 1220 1225 TCA GGT GTT TTA GGT GTG AGA TGT TTC AGT GAA CCA AAG TTC TGA TAC 2112 Ser Gly Val Leu Gly Val Arg Cys Phe Ser Glu Pro Lys Phe * Tyr 1230 1235 1240 CTT GTT TAC ATG TTT GTT TTT ATG GCA TTT CTA TCT ATT GTG GCT TTA 2160 Leu Val Tyr Met Phe Val Phe Met Ala Phe Leu Ser Ile Val Ala Leu 1245 1250 1255 CCA AAA AAT AAA ATG TCC CTA CCA GAA GCC GTT AAA AAA AAA AAA 2205 Pro Lys Asn Lys Met Ser Leu Pro Glu Ala Val Lys Lys Lys Lys 1260 1265 1270 AA 2207 735 amino acids amino acid linear protein 4 Gly Glu Gly Thr Leu * Gly Ile Ala Val His Gly Arg Cys Leu Gln 1 5 10 15 Asp Pro Gly Val Arg * Arg Ala Trp Tyr Cys Ala Leu His Val * 20 25 30 Asp Ala Gln Leu Thr Phe Ala Ala Gly Ala Trp Ile Arg His Phe Leu 35 40 45 Pro Gln Val Leu Leu Asp Ser Pro Asp Thr Ser Leu Cys Asp His Glu 50 55 60 Thr Leu Gly Val Cys Ile Ala Asp Gly Leu Val Trp Cys Pro Glu Leu 65 70 75 80 Cys Ala Gly Arg Ile Leu His Leu Tyr Trp Ala His Asp * Pro Asp 85 90 95 Leu Cys Arg Glu Arg Ala Gly Ala Val Ser Glu Arg Val His Pro Cys 100 105 110 Gly Gly Ser Gln Ala Phe Gln Asp * Glu Leu Gly Gln Gln Asn Gly 115 120 125 Ser Leu Asp * Gln Val Ser Cys * Cys * Gly Leu Pro Gly Ser 130 135 140 Pro Cys Glu Cys Leu Gln Thr Gly Glu Ala Ala Lys His Arg Leu Ala 145 150 155 160 Cys Ala Gly Gly Pro Cys Pro Ala Gly Leu Ser Cys Arg Phe Tyr Arg 165 170 175 Gln Pro Leu Cys Ala Ala Ala Val Leu Pro His * * Gly Arg Asp 180 185 190 Arg Ser Cys Gln Ser Pro Asp Glu Thr Ser Gly His Ile Gln Ala Gly 195 200 205 Pro Arg His Asn Phe Gln Arg Gly Thr Ser Arg Asn Gln Val Pro Gly 210 215 220 Asn Ala Glu Cys Gly * Leu Leu Trp Asp Gly Pro Leu Gly Leu Gln 225 230 235 240 * Arg Gly Leu Leu Ser Tyr Gly Val Val Asp Gly Ala Gly Ala Lys 245 250 255 Ala Ala * Cys Arg Gly Gly Gly His His Asn Gln Val Thr Gly Ala 260 265 270 Gly Leu Pro Thr Leu Cys Cys Leu Pro Val Gly * Ser Ala Pro Cys 275 280 285 Pro Gly Ala His Pro Pro Pro Ala Leu Pro * Pro Lys Pro Arg Thr 290 295 300 Ser Trp Arg Glu Ser Ala Val Leu * Ala Val Ile Gly Gly Arg Glu 305 310 315 320 Arg Lys Asn Val Asn Lys Ser Asp Arg Ser * Ala Ser Asn Pro Arg 325 330 335 Arg His Leu * Glu Ala Cys Gly Leu Pro Ala * Glu Gly Cys Leu 340 345 350 Arg Glu Pro Leu Ser Trp Gly Gly Cys Gln Thr Ser Gln Ala * Thr 355 360 365 Glu Glu Ala Phe Leu * Val Pro Pro Trp Gln Gln Gly Pro Thr Ala 370 375 380 Ala His Cys Pro Leu Gln Arg Gly Gly Arg Val Gly Gln Pro Ala His 385 390 395 400 Arg Gln Val Leu Arg Cys His Val * * Gly Asn Arg Glu Asp Gln 405 410 415 Gly Asp Arg Lys Thr * Thr Cys Thr Ser His Arg Ser * Ser Gln 420 425 430 Glu Arg Ser Pro His Cys Arg Gln Leu Pro Gly Phe Gln Lys Leu Leu 435 440 445 Ala Arg Gly Arg * * Pro Cys Cys Gly Pro Ser Lys Ser Ser Asp 450 455 460 Ala Ala Tyr His Arg Val Asn Ser Lys Asp Cys Arg Ile Val Thr Gly 465 470 475 480 Cys Lys Leu Trp Ser Gly Arg Thr Val * Thr Ala Leu Arg Leu Leu 485 490 495 * Glu * * Ala Arg Tyr Phe Gln Ala Phe Arg Asp Gly Glu Ser 500 505 510 Cys Gly Tyr Phe Leu Lys Leu His Glu * Cys Arg Ser Trp Trp Cys 515 520 525 His Arg Leu Pro * Ser Gly Gly Cys Asn Leu Ala * Glu Gly Tyr 530 535 540 Ser Cys Val Leu Val Gln Pro Leu Ala Glu Arg Gly Arg * Leu Pro 545 550 555 560 Asn Lys Thr Cys Cys Leu Pro Cys Ala Cys Gly Leu Gln Val Gly Leu 565 570 575 Gln * Val Val Pro * Thr Arg Thr Gly Val Leu Glu Thr Leu Trp 580 585 590 Ile Asn Arg Ser * Leu Thr Ser Phe Ser Val Leu Pro Leu Pro Gly 595 600 605 Pro Ser Ala His Val Asn Val Thr Asp Thr Phe Val Cys Ser Phe Val 610 615 620 Cys Ser Tyr Gln Ala Asp Phe Trp Arg Asn Glu Cys Leu Ser Gly Ala 625 630 635 640 Glu Gly Asp His Thr Arg Ala Thr Pro Val * Leu Lys Ser Gln Pro 645 650 655 Phe His Ser Ala Cys Ala Ile Pro Gly Pro Lys Ala Arg Ile Lys Val 660 665 670 Ala Ala Ala Glu Leu Ala Cys * Arg Leu Ala Arg Cys Leu Cys Thr 675 680 685 Ser Gly Val Leu Gly Val Arg Cys Phe Ser Glu Pro Lys Phe * Tyr 690 695 700 Leu Val Tyr Met Phe Val Phe Met Ala Phe Leu Ser Ile Val Ala Leu 705 710 715 720 Pro Lys Asn Lys Met Ser Leu Pro Glu Ala Val Lys Lys Lys Lys 725 730 735 378 base pairs nucleic acid unknown unknown cDNA 5 GGGGAAGGAA CACTGTAGGG GATAGCTGTC CACGGACGCT GTCTACAAGA CCCTGGAGTG 60 AGATAACGTG CCTGGTACTG TGCCCTGCAT GTGTAAGATG CCCAGTTGAC CTTCGCAGCA 120 GGAGCCTGGA TCAGGCACTT CCTGCCTCAG GTATTGCTGG ACAGCCCAGA CACTTCCCTC 180 TGTGACCATG AAACTCTGGG TGTCTGCATT GCTGATGGCC TGGTTTGGTG TCCTGAGCTG 240 TGTGCAGGCC GAATTCTTCA CCTCTATTGG GCACATGACT GACCTGATTT ATGCAGAGAA 300 AGAGCTGGTG CAGTCTCTGA AAGAGTACAT CCTTGTGGAG GAAGCCAAGC TTTCCAAGAT 360 TAAGAGCTGG GCCAACAA 378 486 base pairs nucleic acid unknown unknown cDNA 6 AATGGAAGCC TTGACTAGCA AGTCAGCTGC TGATGCTGAG GGCTACCTGG CTCACCCTGT 60 GAATGCCTAC AAACTGGTGA AGCGGCTAAA CACAGACTGG CCTGCGCTGG AGGACCTTGT 120 CCTGCAGGAC TCAGCTGCAG GTTTTATCGC CAACCTCTCT GTGCAGCGGC AGTTCTTCCC 180 CACTGATGAG GACGAGATAG GAGCTGCCAA AGCCCTGATG AGACTTCAGG ACACATACAG 240 GCTGGACCCA GGCACAATTT CCAGAGGGGA ACTTCCAGGA ACCAAGTACC AGGCAATGCT 300 GAGTGTGGAT GACTGCTTTG GGATGGGCCG CTCGGCCTAC AATGAAGGGG ACTATTATCA 360 TACGGTGTTG TGGATGGAGC AGGTGCTAAA GCAGCTTGAT GCCGGGGAGG AGGCCACCAC 420 AACCAAGTCA CAGGTGCTGG ACTACCTACG CTATGCTGTC TTCCAGTTGG GTGATCTGCA 480 CCGTGC 486 486 base pairs nucleic acid unknown unknown cDNA 7 CCTGGAGCTC ACCCGCCGCC TGCTCTCCCT TGACCCAAGC CACGAACGAG CTGGAGGGAA 60 TCTGCGGTAC TTTGAGCAGT TATTGGAGGA AGAGAGAGAA AAAACGTTAA CAAATCAGAC 120 AGAAGCTGAG CTAGCAACCC CAGAAGGCAT CTATGAGAGG CCTGTGGACT ACCTGCCTGA 180 GAGGGATGTT TACGAGAGCC TCTGTCGTGG GGAGGGTGTC AAACCTCGCA AGCATAGACA 240 GAAGAGGCTT TTCTGTAGGT ACCACCATGG CAACAGGGCC CCACAGCTGC TCATTGCCCC 300 CTTCAAAGAG GAGGACGAGT GGGACAGCCC GCACATCGTC AGGTACTACG ATGTCATGTC 360 TGATGAGGAA ATCGAGAGGA TCAAGGAGAT CGCAAAACCT AAACTTGCAC GAGCCACCGT 420 TCGTGATCCC AAGAGAGGAG TCCTCACTGT CGCCAGCTAC CGGGTTTCCA AAAGCTCCTG 480 GCTAGA 486 540 base pairs nucleic acid unknown unknown cDNA 8 GGAAGATGAT GACCCTGTTG TGGCCCGAGT AAATCGTCGG ATGCAGCATA TCACAGGGTT 60 AACAGTAAAG ACTGCAGAAT TGTTACAGGT TGCAAATTAT GGAGTGGGAG GACAGTATGA 120 ACCGCACTTC GACTTCTCTA GGAATGATGA GCGAGATACT TTCAAGCATT TAGGGACGGG 180 GAATCGTGTG GCTACTTTCT TAAACTACAT GAGTGATGTA GAAGCTGGTG GTGCCACCGT 240 CTTCCCTGAT CTGGGGGCTG CAATTTGGCC TAAGAAGGGT ACAGCTGTGT TCTGGTACAA 300 CCTCTTGCGG AGCGGGGAAG GTGACTACCG AACAAGACAT GCTGCCTGCC CTGTGCTTGT 360 GGGCTGCAAG TGGGTCTCCA ATAAGTGGTT CCATGAACGA GGACAGGAGT TCTTGAGACC 420 TTGTGGATCA ACAGAAGTTG ACTGACATCC TTTTCTGTCC TTCCCCTTCC TGGTCCTTCA 480 GCCCATGTCA ACGTGACAGA CACCTTTGTA TGTTCCTTTG TATGTTCCTA TCAGGCTGAT 540 317 base pairs nucleic acid unknown unknown cDNA 9 TTTTGGAGAA ATGAATGTTT GTCTGGAGCA GAGGGAGACC ATACTAGGGC GACTCCTGTG 60 TGACTGAAGT CCCAGCCCTT CCATTCAGCC TGTGCCATCC CTGGCCCCAA GGCTAGGATC 120 AAAGTGGCTG CAGCAGAGTT AGCTTGCTAG CGCCTAGCAA GGTGCCTTTG TACCTCAGGT 180 GTTTTAGGTG TGAGATGTTT CAGTGAACCA AAGTTCTGAT ACCTTGTTTA CATGTTTGTT 240 TTTATGGCAT TTCTATCTAT TGTGGCTTTA CCAAAAAATA AAATGTCCCT ACCAGAAGCC 300 GTTAAAAAAA AAAAAAA 317 375 base pairs nucleic acid unknown unknown cDNA CDS 1..375 10 GGG GAA GGA ACA CTG TAG GGG ATA GCT GTC CAC GGA CGC TGT CTA CAA 48 Gly Glu Gly Thr Leu * Gly Ile Ala Val His Gly Arg Cys Leu Gln 740 745 750 GAC CCT GGA GTG AGA TAA CGT GCC TGG TAC TGT GCC CTG CAT GTG TAA 96 Asp Pro Gly Val Arg * Arg Ala Trp Tyr Cys Ala Leu His Val * 755 760 765 GAT GCC CAG TTG ACC TTC GCA GCA GGA GCC TGG ATC AGG CAC TTC CTG 144 Asp Ala Gln Leu Thr Phe Ala Ala Gly Ala Trp Ile Arg His Phe Leu 770 775 780 CCT CAG GTA TTG CTG GAC AGC CCA GAC ACT TCC CTC TGT GAC CAT GAA 192 Pro Gln Val Leu Leu Asp Ser Pro Asp Thr Ser Leu Cys Asp His Glu 785 790 795 ACT CTG GGT GTC TGC ATT GCT GAT GGC CTG GTT TGG TGT CCT GAG CTG 240 Thr Leu Gly Val Cys Ile Ala Asp Gly Leu Val Trp Cys Pro Glu Leu 800 805 810 815 TGT GCA GGC CGA ATT CTT CAC CTC TAT TGG TAC GTG CCA ACA GGA CTG 288 Cys Ala Gly Arg Ile Leu His Leu Tyr Trp Tyr Val Pro Thr Gly Leu 820 825 830 TCG TCT CCC TGA CAC CTT GGC TCA CAT GCC ACG GAT GTC TCT GGC TGC 336 Ser Ser Pro * His Leu Gly Ser His Ala Thr Asp Val Ser Gly Cys 835 840 845 AGC TGT TCT CAT TTA GAG TGG GAT AGC CTT AAC ATA CGG 375 Ser Cys Ser His Leu Glu Trp Asp Ser Leu Asn Ile Arg 850 855 860 125 amino acids amino acid linear protein 11 Gly Glu Gly Thr Leu * Gly Ile Ala Val His Gly Arg Cys Leu Gln 1 5 10 15 Asp Pro Gly Val Arg * Arg Ala Trp Tyr Cys Ala Leu His Val * 20 25 30 Asp Ala Gln Leu Thr Phe Ala Ala Gly Ala Trp Ile Arg His Phe Leu 35 40 45 Pro Gln Val Leu Leu Asp Ser Pro Asp Thr Ser Leu Cys Asp His Glu 50 55 60 Thr Leu Gly Val Cys Ile Ala Asp Gly Leu Val Trp Cys Pro Glu Leu 65 70 75 80 Cys Ala Gly Arg Ile Leu His Leu Tyr Trp Tyr Val Pro Thr Gly Leu 85 90 95 Ser Ser Pro * His Leu Gly Ser His Ala Thr Asp Val Ser Gly Cys 100 105 110 Ser Cys Ser His Leu Glu Trp Asp Ser Leu Asn Ile Arg 115 120 125 200 base pairs nucleic acid unknown unknown cDNA CDS 1..198 12 GGC ACA TGA CTG ACC TGA TTT ATG CAG AGA AAG AGC TGG TGC AGT CTC 48 Gly Thr * Leu Thr * Phe Met Gln Arg Lys Ser Trp Cys Ser Leu 130 135 140 TGA AAG AGT ACA TCC TTG TGG AGG AAG CCA AGC TTT CCA AGA TTA AGA 96 * Lys Ser Thr Ser Leu Trp Arg Lys Pro Ser Phe Pro Arg Leu Arg 145 150 155 GGT GTC CTA AGT CCC CAT ACC ATC CTT AGT TGG CCT TCC TTC CCT TCT 144 Gly Val Leu Ser Pro His Thr Ile Leu Ser Trp Pro Ser Phe Pro Ser 160 165 170 GCC CTC AAG GAA CAA GGA AGC CAT CAG GGT GCC TAT AAC ATT AAA CCT 192 Ala Leu Lys Glu Gln Gly Ser His Gln Gly Ala Tyr Asn Ile Lys Pro 175 180 185 TTG AGA GG 200 Leu Arg 190 66 amino acids amino acid linear protein 13 Gly Thr * Leu Thr * Phe Met Gln Arg Lys Ser Trp Cys Ser Leu 1 5 10 15 * Lys Ser Thr Ser Leu Trp Arg Lys Pro Ser Phe Pro Arg Leu Arg 20 25 30 Gly Val Leu Ser Pro His Thr Ile Leu Ser Trp Pro Ser Phe Pro Ser 35 40 45 Ala Leu Lys Glu Gln Gly Ser His Gln Gly Ala Tyr Asn Ile Lys Pro 50 55 60 Leu Arg 65 330 base pairs nucleic acid unknown unknown cDNA CDS 1..330 14 GGG AAT TCT CAC TAG AAA ATT GTC ACA GGT CAA GAC CTA TGT GGG TGG 48 Gly Asn Ser His * Lys Ile Val Thr Gly Gln Asp Leu Cys Gly Trp 70 75 80 ACG CAT TAG TCT TCC TTT TCC TCT GGT TCC ACA GCT GGG CCA ACA AAA 96 Thr His * Ser Ser Phe Ser Ser Gly Ser Thr Ala Gly Pro Thr Lys 85 90 95 TGG AAG CCT TGA CTA GCA AGT CAG CTG CTG ATG CTG AGG GCT ACC TGG 144 Trp Lys Pro * Leu Ala Ser Gln Leu Leu Met Leu Arg Ala Thr Trp 100 105 110 CTC ACC CTG TGA ATG CCT ACA AAC TGG TGA AGC GGC TAA ACA CAG ACT 192 Leu Thr Leu * Met Pro Thr Asn Trp * Ser Gly * Thr Gln Thr 115 120 125 130 GGC CTG CGC TGG AGG ACC TTG TCC TGC AGG ACT CAG CTG CAG GTG AGG 240 Gly Leu Arg Trp Arg Thr Leu Ser Cys Arg Thr Gln Leu Gln Val Arg 135 140 145 GAC GGT GAC GAG GTG CTT GAG TGA GCC CAT ATG TTT GTG TGC TCA TGC 288 Asp Gly Asp Glu Val Leu Glu * Ala His Met Phe Val Cys Ser Cys 150 155 160 CTG GGT TGT TGT GTC TGA GCC TGT CTT GGG TCT GGG TGT TGG 330 Leu Gly Cys Cys Val * Ala Cys Leu Gly Ser Gly Cys Trp 165 170 175 110 amino acids amino acid linear protein 15 Gly Asn Ser His * Lys Ile Val Thr Gly Gln Asp Leu Cys Gly Trp 1 5 10 15 Thr His * Ser Ser Phe Ser Ser Gly Ser Thr Ala Gly Pro Thr Lys 20 25 30 Trp Lys Pro * Leu Ala Ser Gln Leu Leu Met Leu Arg Ala Thr Trp 35 40 45 Leu Thr Leu * Met Pro Thr Asn Trp * Ser Gly * Thr Gln Thr 50 55 60 Gly Leu Arg Trp Arg Thr Leu Ser Cys Arg Thr Gln Leu Gln Val Arg 65 70 75 80 Asp Gly Asp Glu Val Leu Glu * Ala His Met Phe Val Cys Ser Cys 85 90 95 Leu Gly Cys Cys Val * Ala Cys Leu Gly Ser Gly Cys Trp 100 105 110 369 base pairs nucleic acid unknown unknown cDNA CDS 1..369 16 GAG ACC CTC TTT GTG GCT GCC TCT CTG GGT CCC AAG TGG AAT TCT GCC 48 Glu Thr Leu Phe Val Ala Ala Ser Leu Gly Pro Lys Trp Asn Ser Ala 115 120 125 CCT GGA TCA AGG GTA ATC TCT TGT TCT GAC TCT TCA TTT GGA AGG TTT 96 Pro Gly Ser Arg Val Ile Ser Cys Ser Asp Ser Ser Phe Gly Arg Phe 130 135 140 TAT CGC CAA CCT CTC TGT GCA GCG GCA GTT CTT CCC CAC TGA TGA GGA 144 Tyr Arg Gln Pro Leu Cys Ala Ala Ala Val Leu Pro His * * Gly 145 150 155 CGA GAT AGG AGC TGC CAA AGC CCT GAT GAG ACT TCA GGA CAC ATA CAG 192 Arg Asp Arg Ser Cys Gln Ser Pro Asp Glu Thr Ser Gly His Ile Gln 160 165 170 GCT GGA CCC AGG CAC AAT TTC CAG AGG GGA ACT TCC AGG TAA CTC ACC 240 Ala Gly Pro Arg His Asn Phe Gln Arg Gly Thr Ser Arg * Leu Thr 175 180 185 190 ACT CCA GGC GTT GCT GTC CCG CAT GTG TCT CTT TAG TGG CGG GAC AGG 288 Thr Pro Gly Val Ala Val Pro His Val Ser Leu * Trp Arg Asp Arg 195 200 205 TTG GAG CCA CCA CCA ACT TGT GGC CTT TAA CCT CGG GTG CAC CTC TCT 336 Leu Glu Pro Pro Pro Thr Cys Gly Leu * Pro Arg Val His Leu Ser 210 215 220 CTT GGC ACA CCA GTT GTG CTG GAC TCC TCT CCA 369 Leu Gly Thr Pro Val Val Leu Asp Ser Ser Pro 225 230 123 amino acids amino acid linear protein 17 Glu Thr Leu Phe Val Ala Ala Ser Leu Gly Pro Lys Trp Asn Ser Ala 1 5 10 15 Pro Gly Ser Arg Val Ile Ser Cys Ser Asp Ser Ser Phe Gly Arg Phe 20 25 30 Tyr Arg Gln Pro Leu Cys Ala Ala Ala Val Leu Pro His * * Gly 35 40 45 Arg Asp Arg Ser Cys Gln Ser Pro Asp Glu Thr Ser Gly His Ile Gln 50 55 60 Ala Gly Pro Arg His Asn Phe Gln Arg Gly Thr Ser Arg * Leu Thr 65 70 75 80 Thr Pro Gly Val Ala Val Pro His Val Ser Leu * Trp Arg Asp Arg 85 90 95 Leu Glu Pro Pro Pro Thr Cys Gly Leu * Pro Arg Val His Leu Ser 100 105 110 Leu Gly Thr Pro Val Val Leu Asp Ser Ser Pro 115 120 309 base pairs nucleic acid unknown unknown cDNA CDS 1..309 18 GAA CCA AGT ACC AGG CAA TGC TGA GTG TGG ATG ACT GCT TTG GGA TGG 48 Glu Pro Ser Thr Arg Gln Cys * Val Trp Met Thr Ala Leu Gly Trp 125 130 135 GCC GCT CGG CCT ACA ATG AAG GGG ACT ATT ATC ATA CGG TGT TGT GGA 96 Ala Ala Arg Pro Thr Met Lys Gly Thr Ile Ile Ile Arg Cys Cys Gly 140 145 150 155 TGG AGC AGG TGC TAA AGC AGC TTG ATG CCG GGG AGG AGG CCA CCA CAA 144 Trp Ser Arg Cys * Ser Ser Leu Met Pro Gly Arg Arg Pro Pro Gln 160 165 170 CCA AGT CAC AGG TGC TGG ACT ACC TAC GCT ATG CTG TCT TCC AGT TGG 192 Pro Ser His Arg Cys Trp Thr Thr Tyr Ala Met Leu Ser Ser Ser Trp 175 180 185 GTG ATC TGC ACC GTG CCC TGG AGC TCA CCC GCC GCC TGC TCT CCC TTG 240 Val Ile Cys Thr Val Pro Trp Ser Ser Pro Ala Ala Cys Ser Pro Leu 190 195 200 GTA AGG AGA TTC TAG GGG AAG GTA AGA TGG GAA TGG AGA GTG GCA GAG 288 Val Arg Arg Phe * Gly Lys Val Arg Trp Glu Trp Arg Val Ala Glu 205 210 215 GAA CTG CAC TGT GCC TGG CAC 309 Glu Leu His Cys Ala Trp His 220 225 103 amino acids amino acid linear protein 19 Glu Pro Ser Thr Arg Gln Cys * Val Trp Met Thr Ala Leu Gly Trp 1 5 10 15 Ala Ala Arg Pro Thr Met Lys Gly Thr Ile Ile Ile Arg Cys Cys Gly 20 25 30 Trp Ser Arg Cys * Ser Ser Leu Met Pro Gly Arg Arg Pro Pro Gln 35 40 45 Pro Ser His Arg Cys Trp Thr Thr Tyr Ala Met Leu Ser Ser Ser Trp 50 55 60 Val Ile Cys Thr Val Pro Trp Ser Ser Pro Ala Ala Cys Ser Pro Leu 65 70 75 80 Val Arg Arg Phe * Gly Lys Val Arg Trp Glu Trp Arg Val Ala Glu 85 90 95 Glu Leu His Cys Ala Trp His 100 509 base pairs nucleic acid unknown unknown cDNA CDS 1..507 20 TTA GAT GCT GTG AAG GAT GAT GCA CGC ATG CAG GTG AGC TGC TGG GAG 48 Leu Asp Ala Val Lys Asp Asp Ala Arg Met Gln Val Ser Cys Trp Glu 105 110 115 AGA AAC CCT TAC TAC TCT GGT TAG ATG CTG TGA AGG ATG AAT GCA GCA 96 Arg Asn Pro Tyr Tyr Ser Gly * Met Leu * Arg Met Asn Ala Ala 120 125 130 135 TGC AGG TGA GCT GCT CCC AGA GAA ACC CTT ACA GAT AAT TTC TCT AAA 144 Cys Arg * Ala Ala Pro Arg Glu Thr Leu Thr Asp Asn Phe Ser Lys 140 145 150 TGA CCT AAC AGA TGT TTG TGG TTT CCT TTT CCT TCT CAT TCC TTG CAT 192 * Pro Asn Arg Cys Leu Trp Phe Pro Phe Pro Ser His Ser Leu His 155 160 165 TTT CTG ACC CAA GCC ACG AAC GAG CTG GAG GGA ATC TGC GGT ACT TTG 240 Phe Leu Thr Gln Ala Thr Asn Glu Leu Glu Gly Ile Cys Gly Thr Leu 170 175 180 AGC AGT TAT TGG AGG AAG AGA GAG AAA AAA CGT TAA CAA ATC AGA CAG 288 Ser Ser Tyr Trp Arg Lys Arg Glu Lys Lys Arg * Gln Ile Arg Gln 185 190 195 AAG CTG AGC TAG CAA CCC CAG AAG GCA TCT ATG AGA GGC CTG TGG ACT 336 Lys Leu Ser * Gln Pro Gln Lys Ala Ser Met Arg Gly Leu Trp Thr 200 205 210 215 ACC TGC CTG AGA GGG ATG TTT ACG AGA GCC TCT GTC GTG GGG AGG GTG 384 Thr Cys Leu Arg Gly Met Phe Thr Arg Ala Ser Val Val Gly Arg Val 220 225 230 TCA AAC TGG TGA GAT GTG TGA GGG GGC TAG GGT GCC AAA GCT GTG GAC 432 Ser Asn Trp * Asp Val * Gly Gly * Gly Ala Lys Ala Val Asp 235 240 245 CTG GAC TCT GGC CTC TGG GCA GGC AGA TTT GGG GAA GGT GTT CTT TAT 480 Leu Asp Ser Gly Leu Trp Ala Gly Arg Phe Gly Glu Gly Val Leu Tyr 250 255 260 TCT GAG GTA CTT TTC ACG TTT CCC GTT TT 509 Ser Glu Val Leu Phe Thr Phe Pro Val 265 270 169 amino acids amino acid linear protein 21 Leu Asp Ala Val Lys Asp Asp Ala Arg Met Gln Val Ser Cys Trp Glu 1 5 10 15 Arg Asn Pro Tyr Tyr Ser Gly * Met Leu * Arg Met Asn Ala Ala 20 25 30 Cys Arg * Ala Ala Pro Arg Glu Thr Leu Thr Asp Asn Phe Ser Lys 35 40 45 * Pro Asn Arg Cys Leu Trp Phe Pro Phe Pro Ser His Ser Leu His 50 55 60 Phe Leu Thr Gln Ala Thr Asn Glu Leu Glu Gly Ile Cys Gly Thr Leu 65 70 75 80 Ser Ser Tyr Trp Arg Lys Arg Glu Lys Lys Arg * Gln Ile Arg Gln 85 90 95 Lys Leu Ser * Gln Pro Gln Lys Ala Ser Met Arg Gly Leu Trp Thr 100 105 110 Thr Cys Leu Arg Gly Met Phe Thr Arg Ala Ser Val Val Gly Arg Val 115 120 125 Ser Asn Trp * Asp Val * Gly Gly * Gly Ala Lys Ala Val Asp 130 135 140 Leu Asp Ser Gly Leu Trp Ala Gly Arg Phe Gly Glu Gly Val Leu Tyr 145 150 155 160 Ser Glu Val Leu Phe Thr Phe Pro Val 165 480 base pairs nucleic acid unknown unknown cDNA 22 TTAGATGCTG TGAAGGATGA TGCACGCATG CAGGTGAGCT GCTGGGAGAG AAACCCTTAC 60 TACTCTGGTT AGATGCTGTG AAGGATGAAT GCAGCATGCA GGTGAGCTGC TCCCAGAGAA 120 ACCCTTACAG ATAATTTCTC TAAATGACCT AACAGATGTT TGTGGTTTCC TTTTCCTTCT 180 CATTCCTTGC ATTTTCTGAC CCAAGCCACG AACGAGCTGG AGGGAATCTG CGGTACTTTG 240 AGCAGTTATT GGAGGAAGAG AGAGAAAAAA CGTTAACAAA TCAGACAGAA GCTGAGCTAG 300 CAACCCCAGA AGGCATCTAT GAGAGGCCTG TGGACTACCT GCCTGAGAGG GATGTTTACG 360 AGAGCCTCTG TCGTGGGGAG GGTGTCAAAC TGGTGAGATG TGTGAGGGGG CTAGGGTGCC 420 AAAGCTGTGG ACCTGGACTC TGGCCTCTGG GCAGGCAGAT TTGGGGAAGG TGTTCTTTAT 480 29 base pairs nucleic acid unknown unknown cDNA 23 TCTGAGGTAC TTTTCACGTT TCCCGTTTT 29 2121 base pairs nucleic acid unknown unknown cDNA CDS 1..2121 24 TGG CCA TGA GGT GAG TCC AGT GTC TGC AGA CAG CCA GAC TGG GAC CGA 48 Trp Pro * Gly Glu Ser Ser Val Cys Arg Gln Pro Asp Trp Asp Arg 170 175 180 185 GGA TTA GGA CTC ACT CAG CTC AGG GCC TGT TAC TCT GTG CTT TCC AGA 96 Gly Leu Gly Leu Thr Gln Leu Arg Ala Cys Tyr Ser Val Leu Ser Arg 190 195 200 CAC CCC GTA GAC AGA AGA GGC TTT TCT GTA GGT ACC ACC ATG GCA ACA 144 His Pro Val Asp Arg Arg Gly Phe Ser Val Gly Thr Thr Met Ala Thr 205 210 215 GGG CCC CAC AGC TGC TCA TTG CCC CCT TCA AAG AGG AGG ACG AGT GGG 192 Gly Pro His Ser Cys Ser Leu Pro Pro Ser Lys Arg Arg Thr Ser Gly 220 225 230 ACA GCC CGC ACA TCG TCA GGT ACT ACG ATG TCA TGT CTG ATG AGG AAA 240 Thr Ala Arg Thr Ser Ser Gly Thr Thr Met Ser Cys Leu Met Arg Lys 235 240 245 TCG AGA GGA TCA AGG AGA TCG CAA AAC CTA AAG TAG GTG TAC AGT GAG 288 Ser Arg Gly Ser Arg Arg Ser Gln Asn Leu Lys * Val Tyr Ser Glu 250 255 260 265 GCC TTC TCG GGT CAC TGA AGG GGG AAG GTC TTT TTC TCA TCC CCT AGC 336 Ala Phe Ser Gly His * Arg Gly Lys Val Phe Phe Ser Ser Pro Ser 270 275 280 ACT ATG GGT GGT TAG AGT TTG CCC ATC CTA GCC ACC CTT TAT CCA TAT 384 Thr Met Gly Gly * Ser Leu Pro Ile Leu Ala Thr Leu Tyr Pro Tyr 285 290 295 CTA GCA TAG GGC CTA CCT GGA GGG ATA CAG AGA TGC TTC AGA CTC AGC 432 Leu Ala * Gly Leu Pro Gly Gly Ile Gln Arg Cys Phe Arg Leu Ser 300 305 310 CTG ACC TTG TGA GGT TCA TGT GCC AGT GGA AGG AAG GAA CAG GGT AAC 480 Leu Thr Leu * Gly Ser Cys Ala Ser Gly Arg Lys Glu Gln Gly Asn 315 320 325 CAA TGT GGA CAG CCA AGT GCT ATC ATA CAA GGT CAC GTC CTG GGA ACA 528 Gln Cys Gly Gln Pro Ser Ala Ile Ile Gln Gly His Val Leu Gly Thr 330 335 340 345 GGG CTG GGA ACA GGG CAG GTC TAC ACT GGT GTG TCA GTT CAC CTG GTT 576 Gly Leu Gly Thr Gly Gln Val Tyr Thr Gly Val Ser Val His Leu Val 350 355 360 GGG AGA CTG GTG CGT GGG TGA GTT TTT TGG AAA TGT TCC ATA GGA TGC 624 Gly Arg Leu Val Arg Gly * Val Phe Trp Lys Cys Ser Ile Gly Cys 365 370 375 TAT GAA GCT GGG TCC TGT GGA GCT CCT GAG TAG GAC TGT AAA TGA GGT 672 Tyr Glu Ala Gly Ser Cys Gly Ala Pro Glu * Asp Cys Lys * Gly 380 385 390 GAA TGA CTT AGA GGA GAA TGT ATA TCT TTT ATA ATA TTT GGG TCT CTC 720 Glu * Leu Arg Gly Glu Cys Ile Ser Phe Ile Ile Phe Gly Ser Leu 395 400 405 ATC CAA GGG CAT GAC AGG TCT CTC CAT ATC TTT TTA AGT TTT CTT CAT 768 Ile Gln Gly His Asp Arg Ser Leu His Ile Phe Leu Ser Phe Leu His 410 415 420 425 ATA AGC CTT GAA CAT GTC TTA AGT TTA TTC CTT GGT ACT TTC TTT GTT 816 Ile Ser Leu Glu His Val Leu Ser Leu Phe Leu Gly Thr Phe Phe Val 430 435 440 ACT GTT AAT TTA CTT TAT TTC TTC ATT ATT ATT TTA ACT GGT TAC ATT 864 Thr Val Asn Leu Leu Tyr Phe Phe Ile Ile Ile Leu Thr Gly Tyr Ile 445 450 455 ATT TAT TAG TTT ACT ATT ATA TGC CAA ACT ATT GAT TTT ACA AAT ACA 912 Ile Tyr * Phe Thr Ile Ile Cys Gln Thr Ile Asp Phe Thr Asn Thr 460 465 470 TTT CAT AGT AAG AGC TAA TGT TTA CTG AAT TCT TAA CTG TGG CAG GAA 960 Phe His Ser Lys Ser * Cys Leu Leu Asn Ser * Leu Trp Gln Glu 475 480 485 ACT TCT AAG TGC TTA ACA TAT ATA TTA AGT GTT ATG TCA CAG TTA TGA 1008 Thr Ser Lys Cys Leu Thr Tyr Ile Leu Ser Val Met Ser Gln Leu * 490 495 500 505 ACA GCT GCT CAG AAT GAT GTC ACT GTC TCT GTT TTA CCT ATG AAA AAG 1056 Thr Ala Ala Gln Asn Asp Val Thr Val Ser Val Leu Pro Met Lys Lys 510 515 520 CAA ACT CAT ACA GAT TGC AGC TAG TGG TTG AAT TTA CTT ATT TGT TTT 1104 Gln Thr His Thr Asp Cys Ser * Trp Leu Asn Leu Leu Ile Cys Phe 525 530 535 TTG GTT TTA CGT GAT TTC TCT TTG GTT GGG TGG ATA GCA TTA ACA CCT 1152 Leu Val Leu Arg Asp Phe Ser Leu Val Gly Trp Ile Ala Leu Thr Pro 540 545 550 GGA AAT AAG GAA AAT TTT ATT TTC TCC TGA TAC TTG TAG TTC CTT TGT 1200 Gly Asn Lys Glu Asn Phe Ile Phe Ser * Tyr Leu * Phe Leu Cys 555 560 565 TTT TAT AAC CTT ATT GAA TTG CCC AGA ACT TCT AGA GCA TAA TTA CGT 1248 Phe Tyr Asn Leu Ile Glu Leu Pro Arg Thr Ser Arg Ala * Leu Arg 570 575 580 585 AGA ATA GGC ATC CTT GTC TCA TTC CTG AAT TTC CTG GGA AAT TCC TAT 1296 Arg Ile Gly Ile Leu Val Ser Phe Leu Asn Phe Leu Gly Asn Ser Tyr 590 595 600 GGT ATT TAC TGC TAA GAA TGC AGT TGG CTG TTG GTT TTG TAT ATA TGC 1344 Gly Ile Tyr Cys * Glu Cys Ser Trp Leu Leu Val Leu Tyr Ile Cys 605 610 615 CAA AAT TAT TCT TCT GTT TCT AGT TCA TAA AAG ATT TGT TCC CCA TTT 1392 Gln Asn Tyr Ser Ser Val Ser Ser Ser * Lys Ile Cys Ser Pro Phe 620 625 630 GAC ATC TTT CAA AGA GAC CTA TTT GCT GCC ATA TCC CAT CAC TGA TGA 1440 Asp Ile Phe Gln Arg Asp Leu Phe Ala Ala Ile Ser His His * * 635 640 645 TTG GGA GGG AGG ATT TAG CTC GAT TCT CTA TGC TCT GCT CCT AAT AGA 1488 Leu Gly Gly Arg Ile * Leu Asp Ser Leu Cys Ser Ala Pro Asn Arg 650 655 660 665 ATT GTA GGG GCC GAG GTG ACC AGG AGG CCC GAC ACT CAT GGA GAG ACC 1536 Ile Val Gly Ala Glu Val Thr Arg Arg Pro Asp Thr His Gly Glu Thr 670 675 680 TGA AAT AGG TTC CTA TCC TGG CCC CTG GAC CTC ATC TTG GAA CAG CTT 1584 * Asn Arg Phe Leu Ser Trp Pro Leu Asp Leu Ile Leu Glu Gln Leu 685 690 695 TGG CTT GAG GTA CTA GGA CAT CTA GGG CTT TGA GTC AGT GGT TGG CAT 1632 Trp Leu Glu Val Leu Gly His Leu Gly Leu * Val Ser Gly Trp His 700 705 710 CAT CGA TGT GGC TGA GGA AGG GGG CTA GCC AGA TAT ATG GAG AAT GGG 1680 His Arg Cys Gly * Gly Arg Gly Leu Ala Arg Tyr Met Glu Asn Gly 715 720 725 GAC TAG GAC TCC CCT TTC TAC TCA GCT CCA GAG TCC TCC AGG AAA GAA 1728 Asp * Asp Ser Pro Phe Tyr Ser Ala Pro Glu Ser Ser Arg Lys Glu 730 735 740 745 AAC TAC TTT GTT GGT TGT GCC AGG TTT CCT GAG AGA TTC CTT ACC CGT 1776 Asn Tyr Phe Val Gly Cys Ala Arg Phe Pro Glu Arg Phe Leu Thr Arg 750 755 760 TCT TTC AGT TCC AGA CAC TGA GAA CAT TTC TCT GTG CAT GTG TGC ATA 1824 Ser Phe Ser Ser Arg His * Glu His Phe Ser Val His Val Cys Ile 765 770 775 TGT GTA CAC ATG TGT GTG GCT GGC CAC AGG GTA GTG TTA GGA AAA GAT 1872 Cys Val His Met Cys Val Ala Gly His Arg Val Val Leu Gly Lys Asp 780 785 790 ATA TTT GAA TAG AAG CCA TGC AAA GAG CCA AAC AAG GTT GGC AAA CAT 1920 Ile Phe Glu * Lys Pro Cys Lys Glu Pro Asn Lys Val Gly Lys His 795 800 805 GTT TGG CTC TTA ACA TGG CTT CTA TTC AAA GAT AAG CTG ACC CCT CCT 1968 Val Trp Leu Leu Thr Trp Leu Leu Phe Lys Asp Lys Leu Thr Pro Pro 810 815 820 825 TTC CGG AGA CTG TGA GGG ACA GAT GCT ATT CTG GCT TTC AAG TAG AGC 2016 Phe Arg Arg Leu * Gly Thr Asp Ala Ile Leu Ala Phe Lys * Ser 830 835 840 CAA TGA GCT TAA CTT GGC CTG TGG GGA ATG CCT GGC AGC TGT CTG TGG 2064 Gln * Ala * Leu Gly Leu Trp Gly Met Pro Gly Ser Cys Leu Trp 845 850 855 GGG CTC TTG GCC TGC TTT CAA AAT AGC CCT GTC GTT AAA ATG GGA CAG 2112 Gly Leu Leu Ala Cys Phe Gln Asn Ser Pro Val Val Lys Met Gly Gln 860 865 870 CAT CAG TGC 2121 His Gln Cys 875 707 amino acids amino acid linear protein 25 Trp Pro * Gly Glu Ser Ser Val Cys Arg Gln Pro Asp Trp Asp Arg 1 5 10 15 Gly Leu Gly Leu Thr Gln Leu Arg Ala Cys Tyr Ser Val Leu Ser Arg 20 25 30 His Pro Val Asp Arg Arg Gly Phe Ser Val Gly Thr Thr Met Ala Thr 35 40 45 Gly Pro His Ser Cys Ser Leu Pro Pro Ser Lys Arg Arg Thr Ser Gly 50 55 60 Thr Ala Arg Thr Ser Ser Gly Thr Thr Met Ser Cys Leu Met Arg Lys 65 70 75 80 Ser Arg Gly Ser Arg Arg Ser Gln Asn Leu Lys * Val Tyr Ser Glu 85 90 95 Ala Phe Ser Gly His * Arg Gly Lys Val Phe Phe Ser Ser Pro Ser 100 105 110 Thr Met Gly Gly * Ser Leu Pro Ile Leu Ala Thr Leu Tyr Pro Tyr 115 120 125 Leu Ala * Gly Leu Pro Gly Gly Ile Gln Arg Cys Phe Arg Leu Ser 130 135 140 Leu Thr Leu * Gly Ser Cys Ala Ser Gly Arg Lys Glu Gln Gly Asn 145 150 155 160 Gln Cys Gly Gln Pro Ser Ala Ile Ile Gln Gly His Val Leu Gly Thr 165 170 175 Gly Leu Gly Thr Gly Gln Val Tyr Thr Gly Val Ser Val His Leu Val 180 185 190 Gly Arg Leu Val Arg Gly * Val Phe Trp Lys Cys Ser Ile Gly Cys 195 200 205 Tyr Glu Ala Gly Ser Cys Gly Ala Pro Glu * Asp Cys Lys * Gly 210 215 220 Glu * Leu Arg Gly Glu Cys Ile Ser Phe Ile Ile Phe Gly Ser Leu 225 230 235 240 Ile Gln Gly His Asp Arg Ser Leu His Ile Phe Leu Ser Phe Leu His 245 250 255 Ile Ser Leu Glu His Val Leu Ser Leu Phe Leu Gly Thr Phe Phe Val 260 265 270 Thr Val Asn Leu Leu Tyr Phe Phe Ile Ile Ile Leu Thr Gly Tyr Ile 275 280 285 Ile Tyr * Phe Thr Ile Ile Cys Gln Thr Ile Asp Phe Thr Asn Thr 290 295 300 Phe His Ser Lys Ser * Cys Leu Leu Asn Ser * Leu Trp Gln Glu 305 310 315 320 Thr Ser Lys Cys Leu Thr Tyr Ile Leu Ser Val Met Ser Gln Leu * 325 330 335 Thr Ala Ala Gln Asn Asp Val Thr Val Ser Val Leu Pro Met Lys Lys 340 345 350 Gln Thr His Thr Asp Cys Ser * Trp Leu Asn Leu Leu Ile Cys Phe 355 360 365 Leu Val Leu Arg Asp Phe Ser Leu Val Gly Trp Ile Ala Leu Thr Pro 370 375 380 Gly Asn Lys Glu Asn Phe Ile Phe Ser * Tyr Leu * Phe Leu Cys 385 390 395 400 Phe Tyr Asn Leu Ile Glu Leu Pro Arg Thr Ser Arg Ala * Leu Arg 405 410 415 Arg Ile Gly Ile Leu Val Ser Phe Leu Asn Phe Leu Gly Asn Ser Tyr 420 425 430 Gly Ile Tyr Cys * Glu Cys Ser Trp Leu Leu Val Leu Tyr Ile Cys 435 440 445 Gln Asn Tyr Ser Ser Val Ser Ser Ser * Lys Ile Cys Ser Pro Phe 450 455 460 Asp Ile Phe Gln Arg Asp Leu Phe Ala Ala Ile Ser His His * * 465 470 475 480 Leu Gly Gly Arg Ile * Leu Asp Ser Leu Cys Ser Ala Pro Asn Arg 485 490 495 Ile Val Gly Ala Glu Val Thr Arg Arg Pro Asp Thr His Gly Glu Thr 500 505 510 * Asn Arg Phe Leu Ser Trp Pro Leu Asp Leu Ile Leu Glu Gln Leu 515 520 525 Trp Leu Glu Val Leu Gly His Leu Gly Leu * Val Ser Gly Trp His 530 535 540 His Arg Cys Gly * Gly Arg Gly Leu Ala Arg Tyr Met Glu Asn Gly 545 550 555 560 Asp * Asp Ser Pro Phe Tyr Ser Ala Pro Glu Ser Ser Arg Lys Glu 565 570 575 Asn Tyr Phe Val Gly Cys Ala Arg Phe Pro Glu Arg Phe Leu Thr Arg 580 585 590 Ser Phe Ser Ser Arg His * Glu His Phe Ser Val His Val Cys Ile 595 600 605 Cys Val His Met Cys Val Ala Gly His Arg Val Val Leu Gly Lys Asp 610 615 620 Ile Phe Glu * Lys Pro Cys Lys Glu Pro Asn Lys Val Gly Lys His 625 630 635 640 Val Trp Leu Leu Thr Trp Leu Leu Phe Lys Asp Lys Leu Thr Pro Pro 645 650 655 Phe Arg Arg Leu * Gly Thr Asp Ala Ile Leu Ala Phe Lys * Ser 660 665 670 Gln * Ala * Leu Gly Leu Trp Gly Met Pro Gly Ser Cys Leu Trp 675 680 685 Gly Leu Leu Ala Cys Phe Gln Asn Ser Pro Val Val Lys Met Gly Gln 690 695 700 His Gln Cys 705 478 base pairs nucleic acid unknown unknown cDNA 26 TGGCCATGAG GTGAGTCCAG TGTCTGCAGA CAGCCAGACT GGGACCGAGG ATTAGGACTC 60 ACTCAGCTCA GGGCCTGTTA CTCTGTGCTT TCCAGACACC CCGTAGACAG AAGAGGCTTT 120 TCTGTAGGTA CCACCATGGC AACAGGGCCC CACAGCTGCT CATTGCCCCC TTCAAAGAGG 180 AGGACGAGTG GGACAGCCCG CACATCGTCA GGTACTACGA TGTCATGTCT GATGAGGAAA 240 TCGAGAGGAT CAAGGAGATC GCAAAACCTA AAGTAGGTGT ACAGTGAGGC CTTCTCGGGT 300 CACTGAAGGG GGAAGGTCTT TTTCTCATCC CCTAGCACTA TGGGTGGTTA GAGTTTGCCC 360 ATCCTAGCCA CCCTTTATCC ATATCTAGCA TAGGGCCTAC CTGGAGGGAT ACAGAGATGC 420 TTCAGACTCA GCCTGACCTT GTGAGGTTCA TGTGCCAGTG GAAGGAAGGA ACAGGGTA 478 660 base pairs nucleic acid unknown unknown cDNA 27 ACCAATGTGG ACAGCCAAGT GCTATCATAC AAGGTCACGT CCTGGGAACA GGGCTGGGAA 60 CAGGGCAGGT CTACACTGGT GTGTCAGTTC ACCTGGTTGG GAGACTGGTG CGTGGGTGAG 120 TTTTTTGGAA ATGTTCCATA GGATGCTATG AAGCTGGGTC CTGTGGAGCT CCTGAGTAGG 180 ACTGTAAATG AGGTGAATGA CTTAGAGGAG AATGTATATC TTTTATAATA TTTGGGTCTC 240 TCATCCAAGG GCATGACAGG TCTCTCCATA TCTTTTTAAG TTTTCTTCAT ATAAGCCTTG 300 AACATGTCTT AAGTTTATTC CTTGGTACTT TCTTTGTTAC TGTTAATTTA CTTTATTTCT 360 TCATTATTAT TTTAACTGGT TACATTATTT ATTAGTTTAC TATTATATGC CAAACTATTG 420 ATTTTACAAA TACATTTCAT AGTAAGAGCT AATGTTTACT GAATTCTTAA CTGTGGCAGG 480 AAACTTCTAA GTGCTTAACA TATATATTAA GTGTTATGTC ACAGTTATGA ACAGCTGCTC 540 AGAATGATGT CACTGTCTCT GTTTTACCTA TGAAAAAGCA AACTCATACA GATTGCAGCT 600 AGTGGTTGAA TTTACTTATT TGTTTTTTGG TTTTACGTGA TTTCTCTTTG GTTGGGTGGA 660 660 base pairs nucleic acid unknown unknown cDNA 28 TAGCATTAAC ACCTGGAAAT AAGGAAAATT TTATTTTCTC CTGATACTTG TAGTTCCTTT 60 GTTTTTATAA CCTTATTGAA TTGCCCAGAA CTTCTAGAGC ATAATTACGT AGAATAGGCA 120 TCCTTGTCTC ATTCCTGAAT TTCCTGGGAA ATTCCTATGG TATTTACTGC TAAGAATGCA 180 GTTGGCTGTT GGTTTTGTAT ATATGCCAAA ATTATTCTTC TGTTTCTAGT TCATAAAAGA 240 TTTGTTCCCC ATTTGACATC TTTCAAAGAG ACCTATTTGC TGCCATATCC CATCACTGAT 300 GATTGGGAGG GAGGATTTAG CTCGATTCTC TATGCTCTGC TCCTAATAGA ATTGTAGGGG 360 CCGAGGTGAC CAGGAGGCCC GACACTCATG GAGAGACCTG AAATAGGTTC CTATCCTGGC 420 CCCTGGACCT CATCTTGGAA CAGCTTTGGC TTGAGGTACT AGGACATCTA GGGCTTTGAG 480 TCAGTGGTTG GCATCATCGA TGTGGCTGAG GAAGGGGGCT AGCCAGATAT ATGGAGAATG 540 GGGACTAGGA CTCCCCTTTC TACTCAGCTC CAGAGTCCTC CAGGAAAGAA AACTACTTTG 600 TTGGTTGTGC CAGGTTTCCT GAGAGATTCC TTACCCGTTC TTTCAGTTCC AGACACTGAG 660 323 base pairs nucleic acid unknown unknown cDNA 29 AACATTTCTC TGTGCATGTG TGCATATGTG TACACATGTG TGTGGCTGGC CACAGGGTAG 60 TGTTAGGAAA AGATATATTT GAATAGAAGC CATGCAAAGA GCCAAACAAG GTTGGCAAAC 120 ATGTTTGGCT CTTAACATGG CTTCTATTCA AAGATAAGCT GACCCCTCCT TTCCGGAGAC 180 TGTGAGGGAC AGATGCTATT CTGGCTTTCA AGTAGAGCCA ATGAGCTTAA CTTGGCCTGT 240 GGGGAATGCC TGGCAGCTGT CTGTGGGGGC TCTTGGCCTG CTTTCAAAAT AGCCCTGTCG 300 TTAAAATGGG ACAGCATCAG TGC 323 33 base pairs nucleic acid unknown unknown DNA 30 AAGTTGCGGC CGCGAGCATC AGCAAGGTAC TGC 33 25 base pairs nucleic acid unknown unknown DNA 31 TGTCCGGATC CAGTTTGTAC GTGTC 25 

What is claimed is:
 1. A polypeptide comprising a human isoform of the a subunit of prolyl 4-hydroxylase.
 2. The polypeptide of claim 1 wherein the a subunit of prolyl 4-hydroxylase is an α2 subunit.
 3. The polypeptide of claim 2 wherein the amino acid sequence of said polypeptide comprises: (a) the amino acid sequence of SEQ ID NO:3; (b) fragments of the amino acid sequence of SEQ ID NO:3; or (c) amino acid derivatives of the amino acid sequence of SEQ ID NO:3. 